efﬁcient eradication of established tumors in mice with ...corresponding authors:ferry ossendorp,...

Research Article

Efficient Eradication of Established Tumors inMice with Cationic Liposome-Based SyntheticLong-Peptide VaccinesEleni Maria Varypataki1, Naomi Benne1, Joke Bouwstra1,Wim Jiskoot1, andFerry Ossendorp2

Abstract

Therapeutic vaccination with synthetic long peptides (SLP)can be clinically effective against HPV-induced premalignantlesions; however, their efficiency in established malignantlesions leaves room for improvement. Here, we report the hightherapeutic potency of cationic liposomes loaded with well-defined tumor-specific SLPs and a TLR3 ligand as adjuvant. Thecationic particles, with an average size of 160 nm, could stronglyactivate functional, antigen-specific CD8þ and CD4þ T cells andinduced in vivo cytotoxicity against target cells after intradermal

vaccination. At a low dose (1 nmol) of SLP, our liposomalformulations significantly controlled tumor outgrowth in twoindependent models (melanoma and HPV-induced tumors)and even cured 75%–100% of mice of their large establishedtumors. Cured mice were fully protected from a second chal-lenge with an otherwise lethal dose of tumor cells, indicating thepotential of liposomal SLP in the formulation of powerfulvaccines for cancer immunotherapy. Cancer Immunol Res; 5(3);222–33. �2017 AACR.

IntroductionAntigen-specific immunotherapy by vaccination is a promising

approach for the activation of tumor-specific T cells in patients.Intensive research on the application of therapeutic vaccines fortreatment of numerous tumors, including melanoma, lung, andcervical cancers, is intensifying (1–4). Preventive vaccines againstinfections with the high-risk types of HPV have been successfullyintroduced to reduce the mortality associated with human pap-illomavirus (HPV)-induced cervical cancer (5). However, thesevaccines have little effect against established genital lesions, forwhich improved cellular immunity is needed, including HPV-specific CD4þ Th and CD8þ CTLs (6, 7). CD8þ T cells are knownfor their central role in responses to viral infections and cancer;thus, much focus was given to strategies to induce effective T-cell–based immune responses (8), identifying theE6 andE7oncogenicproteins as the most common targets for the development oftherapeutic vaccination against HPV-induced tumors (9, 10).

Immunotherapy using synthetic long peptides (SLP) of theHPV16 E7 oncoprotein efficiently eradicates HPV16þ-established

tumors in mice (11). Patients with high-grade vulvar intraepithe-lial neoplasia vaccinated with a mix of SLPs against E6 and E7proteins have clinical responses, with 47% of them showingcomplete regression of their premalignant lesions after 1 year(12). However, this E6/E7 SLP–based vaccine is not effective inend-stage HPV16þ cervival cancer patients (2). The SLP vaccinesused so far for clinical trials have been emulsified in a clinicalgrade version of incomplete Freund adjuvant, Montanide ISA-51,a suboptimal formulation adjuvant with reported adverse effectssuch as local swelling, pain, redness, and in some cases fever (12).

As an alternative to Montanide, we propose liposomes: lipo-somes are promising as antigen-delivery system in vivo, able toenhance antigen uptake by dendritic cells (DC), and the subse-quent initiation of cell-mediated antitumor immune responsesupon vaccination (13–16). Cationic liposomes are consideredto be more immunogenic than neutral or anionic ones (17, 18).We reported encouraging preclinical results of intradermallyadministered cationic liposomal SLP formulations using modelovalbumin (OVA)-derived SLPs, showing potent in vivo T-cell–priming capacity (19). The direct comparison of these liposomeswith PLGA nanoparticles, Montanide ISA-51, and a squalene-based emulsion formulation revealed the superior capacity ofthese liposomes to raise effective cytotoxic T-cell responses (20).This observation, together with the unfavorable safety profile ofthe currently used adjuvant Montanide ISA-51, makes liposomesattractive candidates as delivery platforms for SLP-based immu-notherapy of cancer.

In this study, we analyzed the potency of our SLP-loadedDOTAP [1,2-dioleoyl-3-(trimethyammonium) propane]–basedcationic formulation as a therapeutic cancer vaccine in twoindependent tumor models. The OVA-derived SLPs containingCTL and Th epitopes (20) were loaded into liposomes, combinedwith different TLR ligands [poly(I:C), Pam3CysK4, CpG], and themost potent formulations were applied in a foreign antigen

1Division of Drug Delivery Technology, Cluster BioTherapeutics, Leiden Aca-demic Centre for Drug Research, Leiden, the Netherlands. 2Department ofImmunohematology and Blood Transfusion, Leiden University Medical Centre,Leiden, the Netherlands.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Authors: Ferry Ossendorp, Department of Immunohematologyand Blood Transfusion, Leiden University Medical Centre, P.O. Box 9600, 2300RC Leiden, the Netherlands. Phone: 317-1526-3843; Fax: 317-1526-5257; E-mail:[email protected]; and Wim Jiskoot, [email protected]

doi: 10.1158/2326-6066.CIR-16-0283

�2017 American Association for Cancer Research.

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(OVA)-expressing melanoma model. In an independent setting,HPV16 E7 SLP was formulated in the same liposomal system andanalyzed as a therapeutic vaccine in the TC-1HPVþ tumormodel.Both formulations were highly effective in the induction ofcellular immunity and tumor control.

Materials and MethodsMaterials

The OVA-derived SLP OVA24 (DEVSGLEQLESIINFEK-LAAAAAK) and the short CTL-epitope peptide OVA8 (SIINFEKL)were produced at the GMP facility of the Clinical Pharmacy andToxicology Department at the Leiden University Medical Center(Leiden, the Netherlands). The OVA17 SLP (ISQAVHAAHAEI-NEAGR), including the OVA Th epitope AAHAEINEA and the21-mer amino-acid long SLP, GQAEPDRAHYNIVTFCCKSDS,harboring the CTL epitope RAHYNIVTF (H-2Db; 49–57) and theTh epitope PDRAHYNIVTF (48–57) of HPV-16 E7 protein, wereproduced in the Immunohematology and Blood TransfusionDepartment of the Leiden University Medical Center (Leiden, theNetherlands). The lipids DOPC (1,2-Dioleoyl-sn-glycero-3-phos-phocholine) andDOTAPwere purchased fromAvanti Polar Lipidsand the TLR ligands (poly(I:C), Pam3CSK4, and CpG) with theirlabeled analogues were obtained from InvivoGen. Carboxyfluor-escein succinimidyl ester (CFSE) was purchased from Invitrogen.Acetonitrile, chloroform, and methanol were obtained from Bio-solve BV and Vivaspin 2 membrane concentrators were purchasedfrom Sartorius Stedim Biotech GmbH. Iscove's modified Dulbec-co'smedium(IMDM; Lonza Verniers) was supplementedwith 8%(v/v) FCS (Greiner Bioscience), 50 mmol/L 2-mercaptoethanol(Sigma-Aldrich), penicillin (100 IU/mL), and 2mmol/L glutamine(Life Technologies). MQ water and 10 mmol/L phosphate buffer(7.7mmol/LNa2HPO4�2H2Oand2.3mmol/LNaH2PO4�2H2O),were filtered through a 0.22-mm Millex GP PES-filter (Millipore)before use. PBS was purchased from B. Braun.

MiceFemale C57BL/6 (H-2b) mice were purchased from Charles

River and congenic CD45.1 (Ly5.1) mice were bred at the LeidenUniversity Medical Center animal facility and used at 8–14 weeksof age according to the Dutch Experiments on Animal Act, whichserves the implementation of "Guidelines on the Protection ofExperimental Animals" by the Council of Europe. All performedanimal experiments were approved by the Animal ExperimentalCommittee of the Leiden University (Leiden, the Netherlands).

Liposome preparationCationic liposomes of DOTAP and DOPC (molar ratio 1:1)

loadedwithOVA24 andOVA17were preparedusing the thin-filmdehydration-rehydrationmethod, as describedpreviously (19, 20).For the liposomes loaded with E7 SLP, the aqueous solution of theSLPs was first adjusted to a pH of about 8.5. For poly(I:C) or CpG-loaded liposomes, the ligand (including 0.5% fluorescently labeledequivalent) was added dropwise to the dispersion in a totalconcentration of 200 mg/mL. For liposomes loaded with thelipophilic Pam3CysK4, the TLR ligandwas dissolved in chloroformtogether with the lipids, before the dry film formation.

Sizing of the liposomes and purification were performed asdescribed previously (19, 20), followed by the particles' physi-cochemical characterization [average diameter (Zave), polydisper-sity index (PDI), zeta-potential determination] as well as the

measurement of their SLP-content (Ultra-Performance LiquidChromatography) and the amount of the loaded TLR ligand(fluorescence measurement in collected nonsolubilized samples;refs. 19, 20).

Vaccination of miceMice were immunized by intradermal injection on the abdom-

inal or tailbase skin area. All formulations were prepared on theday of injection. Vaccination dose was based on the OVA24 or E7SLP concentration, 1nmol (2.5mg forOVA24or 2.3mg for E7 SLP)of peptide in a total volume of 30 mL, and immunizations wereperformed on day 0 (prime immunization) and on day 14 (boostinjection). Vaccinations with adjuvanted liposomes included adose of 0.5–1.0 mg of TLR ligand. During the in vivo studies, bloodsamples were obtained from the tail vein at different time points.

Analysis of antigen-specific CD8þ and CD4þ T-cell responsesby flow cytometry

Staining of the cell surface was performed on blood samplesand splenocytes after red blood cell lysis. Cells were stained instaining buffer for 30 minutes with allophycocyanin (APC)-labeled H-2 Kb-SIINFEKL tetramers or APC-labeled H-2Db -RAHYNIVTF (E749-57) and fluorescently labeled antibodies spe-cific for mouse CD3 (BD Biosciences), CD4, and CD8 (eBios-ciences). 7-Aminoactinomycin D (7AAD; Life Technologies) wasused for the exclusion of dead cells.

Overnight intracellular cytokine analysis of PBMCs was per-formed after incubating the cells with 2 mmol/L of OVA8 and2mmol/L ofOVA17or 2mmol/L of E7 SLP in presence of brefeldinA (7.5 mg/mL; BD Biosciences). The next day the assay wasdeveloped as described (19, 20).

In vivo cytotoxicity assaySplenocytes from na€�ve congenic CD45.1þ mice were labeled

with CFSE for 1 hour at 37�C with either 5 mmol/L (targetpopulation) or 0.5 mmol/L (control) final concentration andpulsed for 1 hour with the short SIINFEKL (OVA SLP; ref. 19) orRAHYNIVTF (E7 SLP) peptide in complete culture medium at37�C. Cells were adoptively transferred intravenously in a 1:1number ratio in recipient previously immunized C57BL/6 miceand two days afterwards (day 24) they were sacrificed and single-cell splenocyte suspensions were analyzed for specific killing (SK)following the equation:

SK ¼ 1�CFSE highCFSE low vaccinated mice

h i

CFSE highCFSE low naive mice

h i8<:

9=;� 100%

Tumor regression experimentB16-OVAmelanoma or TC-1 tumor cells expressing HPV16-E7

proteins were cultured at 37�Cwith 5%CO2 in IMDM containing8% FCS þ 2 mmol/L glutamine and 100 IU/mL penicillin in thepresence of 1 mg/mL for B16-OVA or 400 mg/mL geneticin(G-418) for TC-1 (Life Technologies), nonessential amino acids(10�) (Life Technologies), and 1 mmol/L sodium pyruvate (LifeTechnologies).

On day 0, mice were injected subcutaneously in the flank with1 � 105 tumor cells in 100-mL PBS. On day 9, when tumors werepalpable (>8 mm3) or day 12, when tumors had a large size(>100mm3), mice were split into groups with similar tumor size

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and were vaccinated, as described above. Nonimmunized miceinjected with tumor cells were used as a negative control.

Themice were weighed three times a week and their tumor sizewas measured in three dimensions using a caliper. Mice having atumor size >2,000 mm3 were sacrificed for ethical reasons.

In vivo CD8þ T-cell depletionFor the in vivo CD8þ T-cell depletion, the tumor-bearing mice

were injected intraperitoneally with 100 mg of the mAb, isolatedfrom the 2.43 hybridoma cells that specifically recognizes CD8,on days �1, 1, 7, and 14 after vaccination. The antibody wasprepared and purified as described previously (21).

Statistical analysisThe significance of differences in the in vitro and in vivo assays

were evaluated by GraphPad Prism 6 (GraphPad) software, byusing ANOVA at a 0.05 significance level, followed by Bonferroniposttest. The significance of differences between the survivalcurves was calculated with the log-rank (Mantel–Cox) test.

ResultsCharacterization of liposomes loaded with synthetic peptideantigens and adjuvant

Following the optimized protocol for the preparation ofSLP-loaded liposomes (20), liposomes loaded with OVA- orHPV E7–derived SLPs were produced. Entrapment of SLPs inthe liposomes appeared to be highly dependent on electrostaticinteractions between the liposomes and the peptides (refs.19, 20; Supplementary Table S1; Supplementary Fig. S1). Thetwo-model OVA-SLPs (OVA24 and OVA17) were introducedeither coencapsulated in the same particle or loaded in separateliposomes, and their effect on the induction of antigen-specificT cells was studied, also when combined with defined TLRligands (Supplementary Table S2).

The obtained SLP liposomes were positively charged, rathermonodisperse, and had an average size below 200 nm (Supple-mentary Table S2). The bioactivity of the liposomal-loaded poly(I:C), Pam3CSK4 and CpGwas analyzed in vitro bymeasuring theexpression of the costimulatory marker CD86 on the surface ofDCs incubatedwith the formulations showing that loading of TLRligands in the current liposomes did not diminish their ability toactivate DCs. Also, as shown previously (19), these SLP-loadedcationic DOTAP-liposomes were able to promote DC uptake andprocessing of the SLP antigen for MHC class I presentation,resulting in more efficient activation of SIINFEKL-specificCD8þ T cells (B3Z cells), as compared with the free antigen(Supplementary Fig. S2).

Efficient specific T-cell response induction by liposome–peptide vaccine in vivo

The ability ofOVA SLP–loaded cationic liposome formulationsto induce cell-mediated immune responses in vivo was tested byselecting poly(I:C) as the main TLR ligand adjuvant for a directcomparison with our previous studies (19).

Inmice immunized intradermally twice with themixture of theseparately loaded liposomes (OVA24-lipos. þOVA17-lipos.), ahigh percentage of antigen-specific CD8þ T cells was detected,which was elevated compared with the frequency in mice immu-nized with OVA24-liposomes only, indicating that OVA17-medi-ated help improved the CD8þ response. The response measured

in the blood was significantly higher in mice having been immu-nized with the adjuvant poly(I:C) formulation [OVA24-lipos. þOVA17-lipos. þ poly(I:C); Fig. 1; Supplementary Fig. S3]. Lipo-somes coencapsulated with both SLPs showed increased T-cellnumbers when poly(I:C) was included in the formulation, eithermixedwith the SLP liposomes [OVA24/OVA17-lipos.þpoly(I:C)]or coencapsulated with the peptides (OVA24/OVA17/poly(I:C)-lipos.; Fig. 1).

In addition, all SLP-containing liposomal formulationsinduced high percentages (> 1.0 %) of cytokine-producing CD8þ

T cells and functional cytokine-producing CD4þ T cells weredetected in all groups treated with OVA17-containing liposomalformulations (Supplementary Fig. S4), indicating that OVA17retains its functionality when coencapsulated in liposomes withOVA24.

The most potent formulations presented in Fig. 1 were evalu-ated for their capacity to induce cytotoxic CD8þ T cells that couldkill adoptively transferred target cells in vivo (Fig. 2). The activatedCD8þ T cells in mice immunized with SLP-containing liposomesexhibited strong target cell–specific killing: vaccination with themixture of separate SLP-loaded liposomes gave killing activityabove 80%, andwhen poly(I:C) was included in the formulation,mixed or coencapsulated with the SLPs, the cytotoxic activity waseven further enhanced (Fig. 2).

Similar results were obtained in mice vaccinated with theformulation where the poly(I:C) was replaced by CpG in themixture of the SLPs separately loaded in liposomes (Supplemen-tary Fig. S5) or Pam3CSK4, although the adjuvant effect in thelatter case was less pronounced.

To conclude, our data indicate that the immunogenicity ofSLP-loaded liposomes can be enhanced with the incorporationof different TLR ligands. However, adjuvant formulations withpoly(I:C) tended to be more versatile, because the ligand inthe presence of the TH SLP OVA17 could be either coencapsu-lated with the SLPs or simply mixed before immunizationwithout compromising the strength and the quality of theinduced T-cell response.

Liposome vaccine–mediated regression of establishedmelanoma

The potential of our SLP-liposomal with poly(I:C) formula-tions as therapeutic antitumor vaccines was investigated in anestablished melanoma model (Fig. 3). Nine days after thesubcutaneous B16-OVA tumor cell inoculation, when tumorshad reached a significant palpable size, mice were injectedintradermally twice with the liposome vaccines in a two-weekinterval. Over a period of 60 days, all mice in groups vaccinatedwith poly(I:C)-liposomal formulations could control the out-growth of the established tumors, whereas nonvaccinated miceor mice vaccinated with the free compounds [free OVA24 þOVA17 þ poly(I:C)] had to be sacrificed within three weeksbecause of progressively growing tumors. In detail, mice vac-cinated with the separate OVA24 and OVA17 liposomal mix-tures and poly(I:C) or the poly(I:C)-coencapsulated SLPs(OVA24/OVA17/poly(I:C)-lipos.) showed a substantial tumorregression with a delay of outgrowth of 2 to 4 weeks (Fig. 3). Noadverse side effects were observed at the injection spot. Inaddition, when the OVA24/OVA17/poly(I:C)-lipos. formula-tion was delivered intradermally at the tailbase instead of theabdominal area, a significant 3- to 5-week delay in outgrowthwas observed. Two of 8 mice were even cured of these aggressive

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tumors, indicating that the vaccination effectiveness can bedependent on location and drainage of the injection site.

Effective HPV E7–specific T-cell response induction byliposomes in vivo

Considering the promising results described above, the cationicliposomal formulations loaded with the HPV E7–derived SLPswere tested for their immunogenicity in vivo.

In blood of mice immunized with E7 SLP loaded into lipo-somes (E7-liposomes), a high percentage (ca. 4%) of antigen-specific CD8þ T cells was detected a week after the secondvaccination (day 21; Fig. 4A; Supplementary Fig. S6A). Similar

to the OVA SLP-loaded liposomes, the addition of poly(I:C) inthe formulation led to an even higher number: about 7% of thetotal CD8þ T cells were E7-specific and the adjuvant effect of thepoly(I:C) on the activation of CD8þ T cells was present, regardlessthe way the TLR3 ligand was loaded in the vaccine: coencapsula-tion of the poly(I:C) with the E7 SLP [E7/poly(I:C)-liposomes] ormixed with the E7-liposomes just before the immunization[E7-liposomes þ poly(I:C)] led to an equally strong inductionof T cells. Furthermore, mice immunized with the E7/poly(I:C)-liposomes intradermally on the tailbase, showed a trend to raise ahigher average number of activated T cells, compared with miceimmunized at the abdominal area (Fig. 4A). Ten days after the

Figure 2.

In vivo cytotoxicity assay. The mean percentages of thekilling activity of OVA formulations are presented on thebasis of the ratio of differentially labeled, transferredCD45.1þ specific target cells, which could be detected insplenocytes of mice immunized with 1 nmol of OVASLPs-loaded formulations. Bar graph shows the meanpercentages of cell killing (þSEM) on day 24. Dataevaluated by one-way ANOVA with Bonferroni multiplecomparison test; �� , P < 0.0001. Representativehistograms of CFSE-labeled target cells: right peak,peptide pulsed; left peak, control.

Figure 1.

Antigen (SIINFEKL)-specific CD8þ T-cell responses in blood (day 21) and in splenocytes (day 24) following intradermal immunization with 1 nmol ofOVA24 SLP in different formulations on day 0 and 14. OVA-specific CD8þ T-cell responses were detected with SIINFEKL-H-2 Kb tetramers. The experimentwas performed twice with comparable results, which were evaluated by one-way ANOVAwith Bonferroni multiple comparison test; � , P < 0.05. Each dot representsthe response of an individual mouse (n ¼ 5, mean � SEM).






boost immunization (day 24), the number of the CD8þ T cellswas analyzed also in spleens of immunized mice, showing againsignificantly higher frequencies of antigen-specific T cells for thegroups having received adjuvantþ liposomal vaccines, compared

with the ones that received the vaccine of the free compounds[free E7 þ poly(I:C); Fig. 4B].

Analysis of blood samples and splenocytes with the H-2Db–

RAHYNIVTF tetramer antibody revealed the potency of the

Figure 3.

Tumor outgrowth in a therapeutic melanoma model, in mice immunized intradermally (i.d.) twice (as the arrows indicate) with different OVA SLPs formulations,after subcutaneous injection on day 0 with 1 � 105 B16-OVA cells. Tumor sizes in individual mice (n ¼ 8; A) and survival plots (left: i.d. abdominal skinvaccinations, right: tailbase i.d. vaccination; B) are presented. The curve of the two OVA SLPsþ poly(I:C) liposomal formulations are significantly different from thefree OVA SLPs þ poly(I:C), �� , P < 0.0001 calculated with the log-rank (Mantel–Cox) test. The experiment was performed twice with comparable results.

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Figure 4.

E7-specific CD8þ T-cell responses in blood (day 21; A) and splenocytes (day 24; B) upon intradermal immunization of five mice per group (at the abdominalarea or tailbase) with 1 nmol of E7 SLP on day 0 and 14. Frequencies of E7-specific CD8þ T cells were detected with RAHYNIVTF-MHC Db tetramer. The experimentwas performed twice with comparable results. Intracellular cytokine analysis in blood of immunized mice at day 21 (C–F). Blood samples were stimulatedex vivo overnight with the E7 SLP. Plots showCD8þ T cells producing IFNg (C); CD8þ T cells producing both IFNg and TNFa (D); CD4þ T cells producing IFNg (E); andCD4þ T cells producing IL2 (F). Data shown are averages of 5 mice per group þ SEM and were evaluated by one-way ANOVA with Bonferroni multiplecomparison test; � , P < 0.05; �� , P < 0.001; and ��, P < 0.0001.






vaccines in the expansion of primed antigen-specific CD8þ T cells,while their functionality was analyzed by cytokine production.Because the E7 SLP vaccine also contains a Th epitope, weanalyzed the induction of E7-specific CD4þ T cells after ex vivorestimulation of blood samples from immunized mice collectedon day 21 (1 week after the boost immunization).

The intracellular cytokine analysis showed that all liposomalformulations induced high numbers of IFNg-producingCD8þ T cells, above 3 %, which correlates with the data of theRAHYNIVTF -tetramer analysis. This percentage showed a 2-foldincrease when poly(I:C) was coencapsulated with the SLP [E7/poly(I:C)-liposomes], but notwhenmixedwith the E7-liposomes[E7-liposomes þ poly(I:C); Fig. 4C]. Furthermore, E7-liposomevaccination induced activation of significant numbers of func-tional E7-specific CD4þ T cells, producing IFNg (Fig. 4E; Supple-mentary Fig. S6B). Activation of cytokine-producing CD4þ T cellsunderlines the development of a Th1-oriented response, charac-terized by the secretion of IFNg and IL2 cytokines (Fig. 4F). Withrespect to that, a fraction of IFNg-producing CD8þ T cells alsoproduced the effector cytokine TNFa (Fig. 4D), indicating thatspecific CD4þ T cells were not only expanded by the vaccination,but also produced different functional type I cytokines, furtheramplifying the effector T-cell immune response.

As also shown for theOVA SLP–loaded liposomes, intradermalimmunization of mice with 1 nmol of E7 SLP-loaded into lipo-somes induced strong in vivo killing, above 70%, in contrast tovaccination with free E7 (Fig. 5). Addition of poly(I:C) to theliposomal formulation [E7-liposomesþ poly(I:C)], however, didnot further enhance cytotoxic activity. Vaccination with the E7SLP/poly(I:C) coencapsulated liposomal formulation inducedcytotoxicity up to 80% (Fig. 5).

In conclusion, the poly(I:C)–E7-liposome formulations effi-ciently induced a functional CD8 and CD4 T-cell–mediatedimmune responses. The E7 SLP–specific CD8þ T-cell frequencyinduced by the liposomes showed a significant increase of theT-cell percentage as compared with the free E7 SLP vaccines, withhigh in vivo killing capacity.

Adjuvant–E7 peptide–loaded liposomes efficiently eradicateestablished HPV16þ tumors

The therapeutic potency of the poly(I:C)–E7 SLP-liposomevaccines [containing 2.3 mg E7 SLP and 1 mg poly(I:C)] wasinvestigated in mice bearing (s.c.) tumors induced by the TC-1cell line, expressing HPV16 E7 protein. As a control, the freecompounds [free E7 SLPþ poly(I:C)] were used as vaccines in thesame doses in PBS and emulsified in Montanide. The liposomalformulations were directly compared with the clinical "goldstandard" Montanide ISA-51 mixed with a 65-fold higher doseof E7 SLP and a 20-fold higher dose of poly(I:C) (high dose inMontanide: 150 mg and 20 mg, respectively; ref. 21).

Therapeutic treatment started 9 days after TC-1 tumor cellsinoculation when mice had clearly palpable tumors. Nontreatedmice, mice vaccinated with the free compounds vaccine [freeE7 þ poly(I:C)], or mice injected with the low dose of vaccine inMontanide had to be sacrificed within 3 weeks because ofprogressive tumor growth (Fig. 6). Within a period of 130 days,all mice in groups vaccinated once with the poly(I:C)–E7liposomal formulations or the standardized (high) dose inE7-Montanide ISA-51 emulsion, could control outgrowth ofthe established TC-1 tumors. Mice vaccinated intradermallywith the E7/poly(I:C)-liposomes showed a substantial tumorregression with 5 of 7 mice being completely cured from their

Figure 5.

In vivo cytotoxicity against RAHYNIVTF-presenting transferred target cells. The mean percentages of the killing activity of E7 formulations are presented onthe basis of the frequency of the transferred CD45.1þ specific target cells that could be detected in splenocytes of mice immunized with 1 nmol of E7 SLPs-loadedformulations. Bar graph shows the mean percentages (þ SEM) on day 24. Representative histograms of CFSE-labeled target cells: right peak, peptidepulsed; left peak, control. Data evaluated by one-way ANOVA with Bonferroni multiple comparison test, �� P < 0.0001.

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established tumors. When the same formulation was intrader-mally administered at the tailbase instead of the abdominalarea, 100% of the mice eradicated their tumors and were fullyprotected till the end of the experiment (Fig. 6).

Mice vaccinated with 2.3 mg of E7 SLP in nonadjuvantedliposomes (E7-liposomes) showed a delay on the tumor out-

growth, comparable with that observed in mice injected with a65-fold higher dose of E7 SLP emulsified in Montanide ISA-51and combined with a higher dose of poly(I:C) (Fig. 6; Supple-mentary Fig. S7A and S7B).

We observed a strong administration route–dependenteffect on regression of tumor outgrowth, with the intradermal

Figure 6.

Adjuvanted E7-liposomes efficiently eradicate established E7-specific tumors. Graphs depict tumor outgrowth in a therapeutic model, in mice immunizedintradermally (i.d.) or subcutaneously (s.c.) at day 9 after tumor inoculation (first arrow), with different E7 SLP formulations, after they were subcutaneously injectedon day 0 with 1 � 105 TC-1 cells. Tumor sizes in individual mice (A) and survival plots (B) are presented. The curve of the E7/poly(I:C)-liposomes i.d.tailbase is significantly different from the one of free E7þ poly(I:C) high dose in Montanide; �� . P < 0.005 evaluated with the log-rank (Mantel–Cox) test. Theexperiment was performed twice with comparable results. Light gray arrow in both A and B plots indicate the tumor-rechallenge time point (D85).






vaccination being more efficient than the subcutaneous one(Fig. 6; Supplementary Fig. S7A and S7B). In addition,although no significant difference was observed between micevaccinated intradermally at the abdominal area or the tailbase,when mice were injected subcutaneously the different areassignificantly influenced the tumor regression (SupplementaryFig. S7A and S7B). Similarly, when the E7/poly(I:C)-liposomalformulation was delivered subcutaneously, the tailbase injec-tion seemed to be more potent than the abdominal one (50%and 12.5% cured mice, respectively), suggesting that vaccina-tion effectiveness might depend not only on the administra-tion route, but also the injection site, as also observed in theB16-OVA model (Fig. 3).

In all mice vaccinated with liposomal formulations, no adverseside effect was observed at the injection spot, irrespective of theadministration route or site; in contrast, the high-dose of Mon-tanide-induced skin irritation on the shaved tailbase area of theimmunized mice.

Tumor rechallenge on day 85 in mice vaccinated with E7/poly(I:C)-liposomes (i.d. abdominal area/tailbase and s.c. tailbase)showed that all intradermally vaccinated mice that were cured oftheir initial tumors were also fully protected by the new tumorinoculation. This observation suggested that these mice raisedfunctional memory T cells to control newly injected tumor cells(Fig. 6; Supplementary Fig. S7C), as confirmed by high frequen-cies of functional antigen-specific CD8þ T cells detected in bloodat this time point (data not shown).

Finally, to be able to attribute the tumor regression to theinduction of a E7 tumor–specific T-cell–mediated immunity,mice depleted from CD8þ T cells were vaccinated with theE7/poly(I:C)-liposomal formulation via the most potent admin-istration routes (i.d. abdominal area/tailbase). All mice that wereCD8þ T-cell–deficient had to be sacrificed within three weeks dueto their tumors' size reaching the humane endpoint (Fig. 7A;Supplementary Fig. S7D), showing the crucial role of CD8þ

effector T cells in this system.Nondepleted mice with very large tumors (>400 mm3) treated

intradermally with the E7/poly(I:C)-liposomes, either at theabdominal area or at the tailbase, showed a remarkable tumorregression in this experiment (Fig. 7B). In detail, 75%mice treatedintradermally with one shot of E7/poly(I:C)-liposomes werecured from their established tumors. Even when their tumorshad reached a size of about 500 mm3, a single vaccination couldstill induce full regression of large tumor masses, pointing to theimportance of the administration route effect and the potential ofthese cationic liposomes as a therapeutic cancer vaccine system forSLPs.

DiscussionHere, the concept of cationic liposome-based formulations was

investigated as the backbone for improved delivery of SLPs har-boring defined tumor antigens for active immunotherapy. SLPshave gained much interest not only because of successful clinical

Figure 7.

Tumor eradication is dependent on CD8þ T-cell immunity. Survival of WT mice immunized i.d. (arrow) with the E7/poly(I:C) formulation, after having beensubcutaneously injected on day 0 with 1 � 105 TC-1 cells, is compared with WT mice with depleted CD8þ T cells (A). Eradication of established large tumors wasobserved in WT mice immunized with the E7/poly(I:C) formulation intradermally on the abdominal area and tail base (B).

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trials with overlapping long peptides in HPV-induced premaligantlesions, but also the importance of mutated antigenic sequences(neoepitopes) in several tumor types that can be readily producedas SLPs covering themutated area (22). This opens the opportunityfor personalized specific immunotherapy, but it requires optimalformulation for multiple and diverse SLP sequences.

We showed previously that a poly(I:C)–liposomal formulationinduced a strong and long-lasting cytotoxic T-cell–mediatedimmune response against OVA24, an SLP antigen with relativelylow immunogenicity (19), andwe reported the superior efficiencyof liposomes when compared with other SLP-loaded particulatesystems (20). Even though the complexes of the MHC class I þpeptide can be recognized by na€�ve CD8þ T cells directly, tobecome effector and cytotoxic T cells, they require sufficientcostimulatory signals from the activated DCs (23).

It is known that particulate delivery systems, includingliposomes, offer numerous advantages for DC-targeted vacci-nation in comparison with free antigens (24). The enhancedimmunogenicity observed for liposomal SLPs as comparedwith free SLPs, is likely due to enhanced cellular uptake andthe capability of the DOTAP-based liposomes to induce acti-vation and maturation of DCs (19, 25). As the developedformulation does not include any targeting molecule or device,the DCs are most likely targeted in a passive way, for instancedue to the small size and/or the cationic nature of the lipo-somes. Moreover, as shown in the current study, cationicliposomes are excellent carriers for defined TLR ligands, whichretain their DC-maturing capacity after particle uptake. Regard-ing the liposomes' size, it has been suggested that small par-ticles (ca. 200 nm) are naturally taken up by endocytosis,resulting in a cellular immune response through DC targeting,whereas larger particles are more likely to be phagocytosed bymacrophages, leading to a humoral immune response (26, 27).However, some reported results are contradictory and theimmunogenicity of liposomal vaccines depends on multipleother factors (such as administration route) that need to betaken into account (28).

Encouraging findings have been reported regarding the precisetargeting of DCs upon intravenous administration of nonfunc-tionalized RNA-lipoplexes (29). In the current study, using arather low dose of OVA or E7-derived long peptide (�2.5 mgSLP/mouse) into cationic DOTAP-based liposomes, we reporthigh frequencies of SLP-specific CD8þ T cells as well as functionalCD8þ and CD4þ T-cell responses inmice, confirming the potencyof positively charged liposome formulations. The way thatOVA-derived peptides, OVA24 and OVA17, were loaded into theliposomal formulation (coencapsulated or delivered in separateparticles) did not compromise the induced immune response.Finding suggested that multiple liposomes are most likely takenup by the same DC. Future confocal microscopy studies usingfluorescently labeled liposomes and labelled SLP could givemoreinsight into the fate of the liposomes and the antigen followingadministration in vivo.

Activation and proliferation of CD4þ T cells is crucial for anoptimal CD8þ T-cell response and development of memory (4),as CD4þ T cells help in the differentiation of na€�ve CD8þ T cellsinto effector T cells through activation signals, mostly IL2 secre-tion (30). Enhanced production of IFNg could also contribute toanti-HPV immunity (31), as IFNg-associated T-cell responses areweak or absent in patients with cervical cancer (32). So, based onthe robust CD4þ T-cell responses detected, the superiority of the

liposomes over free SLPs as a cancer therapeutic vaccine can beshown.

Although the use of a Th SLP significantly improved CD8þ

T-cell priming (33), the inclusion of a TLR ligand adjuvantappeared to be crucial for the control of the tumor outgrowth.In both humans andmice, a large list of TLR ligands is known andall of them function as adjuvants, stimulating DC targeting andactivation (25) and cross-presentation (23). In this study, theTLR3 ligand poly(I:C) was the adjuvant most frequently com-bined with the liposomes; however, replacement of poly(I:C)with the TLR9 agonist CpG and the TLR1/2 agonist Pam3CSK4,which can provide different danger signals, was also achieved.Noteworthy, in contrast to several studies in which codelivery ofthe adjuvant with the antigen was beneficial (24, 34), we showedthat the adjuvant effect of poly(I:C) was independent of the way itwas formulated: the coencapsulation of poly(I:C) with the SLP inliposomes or themixture of the ligandwith the SLP-liposomes areboth strongly immunogenic vaccines. Altogether, the results onthe OVA-loaded liposomes' characterization indicate not onlythat is feasible to load two SLPs, either separately or coencapsu-lated into liposomes, suggesting that DCs can engulf multipleliposomes simultaneously, but also that the TLR ligands can bereplaced and the peptide ratios can be adjusted to the desired finalconcentration, for better control and easier adjustment of peptideratios in multiple long-peptide vaccines.

The route of immunization is another crucial factor that cangreatly influence the nature of the induced T-cell response (35),considering the different cell types that can be targeted by justchanging the vaccine delivery route. Although many administra-tion routes are under investigation, the most commonly usedroute remains the subcutaneous (s.c.) one. Here we showed that asingle intradermal tailbase vaccination of TC-1 tumor-bearingmice with a low dose of E7/poly(I:C)-liposomes led to completeclearance of the tumors in 100% of the mice. Upon intradermalvaccination, antigen presentation depends on the targeted APCsthat transport the injected antigen towards the draining lymphnodes (36) or the recruitment of DCs subsets in the local tissue orthe lymph nodes for promoting the cross-presentation of theantigen (37). Considering this, attractive cellular target for vacci-nation approaches canbe theCD8aþDCs and their equivalents intissues, due to their enhanced ability to present exogenous antigenonMHC class I and their strong capacity to provide a robust signal3 (38). However, the contribution of skin-resident DCs to theliposomes' immunogenicity cannot be excluded.

Cationic liposomes formulated with the appropriate TLRligand and SLPs may be well-suited to optimally target the mostefficient APC via the intradermal route. Our results showingadequate tumor eradication obtained with two independent SLPmodel systems using two aggressive tumor types (TC-1 and B16melanoma), suggest that cationic liposomes are not only anexcellent delivery platform for peptide-based therapeutic cancervaccines, but also superior to the clinically usedMontanide ISA-51emulsion or polymeric particulate delivery system, as also shownin our recent comparative study (20). With respect to the latter,polymeric pLHMGA particles loaded with a longer version ofthe HPV16 E7-derived SLP showed a prolongation of mice sur-vival (3 weeks) when used as therapeutic vaccine in the TC-1model (39); however, no tumor eradication was observed evenwhen a high dose (i.e., 43-fold higher) of E7-loaded particles (100SLP mg/mouse) combined with poly(I:C) (50 mg/mouse, i.e.,50-fold higher than the dose in the current study) was injected






subcutaneously on the flank of tumor-bearing mice. RegardingtheMontanide ISA-51 emulsion, the exact adjuvantmechanism isnot well understood; however, it is believed that it functions via asustained release from the local antigen depot. Considering thegreat efficiency of our liposomes, we could conclude that inter-nalization of particles may be more crucial for the induction of acellular immune response than the formation of a depot, in linewith our recent observations with PLGA particles (40).

Combination of liposomes for immunotherapy with othertherapeutic strategies, such as chemotherapy [particles loadedwith doxorubicin, cisplatin (41, 42), or paclitaxel (43)] or pho-todynamic therapy (PDT) can be expected to lead to better thanthe reported in literature result (44).

In conclusion, adjuvant–cationic liposomes loaded with SLPscan be an efficient monotherapy against established tumors inmice. SLP/poly(I:C)-loaded liposomes, when administered intra-dermally, induce a strong T-cell–mediated immunity, able tocontrol the tumor outgrowth, as we showed in two independentmodels (B16-OVA and TC-1). Poly(I:C)-adjuvanted E7-loaded-liposomes cured 75%–100% of the immunized mice from theirlarge established tumors and they were proved to be moreeffective than the clinically used Montanide formulation loadedwith the standardized 65-fold and 20-fold higher dose of E7 SLPand poly(I:C), respectively. The liposomes' versatility regardingthe loading of different antigens and TLR ligands makes them apromising platform for SLP-based immunotherapy of cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: E.M. Varypataki, J. Bouwstra, W. Jiskoot, F. OssendorpDevelopment of methodology: E.M. Varypataki, N. Benne, F. OssendorpAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.M. Varypataki, N. Benne, F. OssendorpAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.M. Varypataki, N. Benne, W. Jiskoot, F. OssendorpWriting, review, and/or revision of themanuscript: E.M. Varypataki, N. Benne,J. Bouwstra, W. Jiskoot, F. OssendorpAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.M. Varypataki, F. OssendorpStudy supervision: E.M. Varypataki, J. Bouwstra, W. Jiskoot, F. Ossendorp

Grant SupportThis study was financially supported by the funding organization, "Leidse

profileringsgebied Translational Drug Discovery and Development en desamenwerking LACDR en LUMC" and a CRI Clinic and Laboratory IntegrationProgram (CLIP) Grant (to F. Ossendorp).

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 October 20, 2016; revised January 10, 2017; accepted January 10,2017; published OnlineFirst January 31, 2017.

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2017;5:222-233. Published OnlineFirst January 31, 2017.Cancer Immunol Res Eleni Maria Varypataki, Naomi Benne, Joke Bouwstra, et al. Liposome-Based Synthetic Long-Peptide VaccinesEfficient Eradication of Established Tumors in Mice with Cationic

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