nanoparticle conjugation of human papillomavirus 16 e7...

Research Article

Nanoparticle Conjugation of HumanPapillomavirus 16 E7-long Peptides EnhancesTherapeutic Vaccine Efficacy against SolidTumors in MiceGabriele Galliverti1,2, M�elanie Tichet2, Sonia Domingos-Pereira3, Sylvie Hauert1,4,Denise Nardelli-Haefliger3, Melody A. Swartz1,4,5, Douglas Hanahan2, andStephan Wullschleger2

Abstract

Treatment of patients bearing human papillomavirus(HPV)-related cancers with synthetic long-peptide (SLP) ther-apeutic vaccines has shown promising results in clinical trialsagainst premalignant lesions, whereas responses against laterstage carcinomas have remained elusive. We show that con-jugation of a well-documented HPV-E7 SLP to ultra-smallpolymeric nanoparticles (NP) enhances the antitumor efficacyof therapeutic vaccination in different mousemodels of HPVþ

cancers. Immunization of TC-1 tumor-bearing mice with asingle dose of NP-conjugated E7LP (NP-E7LP) generated alarger pool of E7-specific CD8þ T cells with increased effectorfunctions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8þ T cells that wasnot accompanied by concomitant accumulation of regulatoryT cells (Tregs), resulting in a higher CD8þ T-cell to Treg ratio.

Consequently, the amplified immune response elicited by theNP-E7LP formulation led to increased regression of large,well-established tumors, resulting in a significant percentage ofcomplete responses that were not achievable by immunizingwith the non-NP–conjugated long-peptide. The partialresponses were characterized by distinct phases of regression,stable disease, and relapse to progressive growth, establishinga platform to investigate adaptive resistance mechanisms. Theefficacy of NP-E7LP could be further improved by therapeuticactivation of the costimulatory receptor 4-1BB. This NP-E7LPformulation illustrates a "solid-phase" antigen delivery strat-egy that is more effective than a conventional free-peptide("liquid") vaccine, further highlighting the potential of usingsuch formulations for therapeutic vaccination against solidtumors. Cancer Immunol Res; 6(11); 1301–13. �2018 AACR.

IntroductionHuman papillomaviruses (HPV) are the most common sexu-

ally transmitted agents worldwide, and it is estimated thatmost ofthe world population will come in contact with these viruses atleast once in their lifetime (1). Chronic infection with oncogenicHPVs can lead to the development of asymptomatic neoplasias inthe cervix and vulva and in the head and neck region (1–3). In

some patients, these lesions eventually progress to symptomaticsquamous cell carcinomas (1).

In contrast tomany types of cancer, for which finding a suitableantigen for therapeutic vaccines might require complex analysesof the tumor and prediction of neoantigen binding toMHC(4, 5),HPVþ malignancies can be targeted by using the viral oncopro-teins (6). Clinical trials using HPV oncoprotein–derived syntheticlong-peptide (SLP) vaccines to treat patients with early-stage vulvar and cervical intraepithelial neoplastic lesions haveshown promising results, in rare cases leading to completeresponses (7–9). However, SLP vaccines have poor efficacy inmost such patients, as well as in those with malignant disease(7, 10). Clinical responses to SLP vaccines are dependent ondendritic cells (DC) that process SLPs into short peptides forpresentation on MHC molecules (11–13) to elicit CD8þ T-cellresponses (7, 9). Although SLPs can elicit a strong CD4þ type 1 Thelper (Th1) response in patients treated with HPV vaccines, theinduced CD8þ T cell counterpart is usually weaker (7–9, 14). Inaddition to themagnitude of the CD8þ T-cell response, the CD8þ

T-cell/regulatory T-cell (Treg) ratio is a prognostic marker forHPVþ cancers (15). Therapeutic vaccination of HPVþ cancerpatients typically leads to an increased abundance of Tregs(16), and larger premalignant lesions show a greater increase inintratumoral Tregs compared with smaller ones (7). The stimu-lation of Treg infiltration consequent to therapeutic vaccination issuspected to contribute to the poor efficacy of SLP vaccines (17).As such, identifying vaccine formulations, which both enhance

1Institute of Bioengineering, Swiss Federal Institute of Technology Lausanne(EPFL), Lausanne, Switzerland. 2Swiss Institute for Experimental CancerResearch, School of Life Sciences, EPFL, Lausanne, Switzerland. 3UrologyResearch Unit, University Hospital of Lausanne (CHUV), Lausanne, Switzerland.4Institute forMolecular Engineering, University of Chicago, Chicago, Illinois. 5TheBen May Department for Cancer Research, University of Chicago, Chicago,Illinois.

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

Corresponding Authors: Stephan Wullschleger, Swiss Federal Institute ofLausanne (EPFL), Station 19, Lausanne CH-1015, Switzerland. Phone: 412-1693-0655; Fax: 412-1693-0660; E-mail: [email protected]; DouglasHanahan, [email protected]; and Melody A. Swartz, Eckhardt ResearchCenter, Room379, 5640South Ellis Avenue, Chicago, IL 60637. Phone: 773-702-0452; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-18-0166

�2018 American Association for Cancer Research.

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recruitment of activated CTLs while avoiding the concomitantinduction of Tregs, may be necessary for efficacy against cervicaland other HPVþ malignancies.

Bioengineering technologies are proving applicable to immu-notherapy, for example, the development of lymph node–targeting vaccines (18–21), including an ultra-small polymericnanoparticle (NP) formulation can boost immune responses tomodel tumor antigens (18). The chemical structure of this 30-nmdiameter NP, with disulfide bonds on the surface, enables loadingwith thiol-bearing antigenic payload molecules (22). Their smallsize enables theseNPs to rapidly reach draining lymphnodes aftersubcutaneous and intradermal administration, where they can beefficiently taken up by DCs (22). Once internalized by DCs, thedisulfide bonds that bind the antigen payload to the surface of theNPs are broken, and the released antigen is preferentially pro-cessed and loaded onto MHC-I molecules to activate the CD8þ

T-cell response (18, 22, 23).We hypothesized that NP conjugation of SLPs derived from

the HPV protein E7 (E7LP) would boost immune responsesagainst E7-expressing neoplasias, in particular, well-estab-lished solid tumors in different anatomical locations. To testthis hypothesis, the E743-77 SLP (E7LP; ref. 24) was conjugatedto NPs, and compared with the non–NP-conjugated free(liquid) E7LP in several mouse models of HPVþ cancers. Wecomparatively evaluated efficacy of the two vaccine formula-tions in mice bearing tumors derived from inoculating HPV16E6/7–transformed TC-1 cells, which have been widely used totest therapeutic strategies against HPVþ cancers (24–28), anda new squamous cell carcinoma cell line (termed SC1) derivedfrom a skin tumor in an HPV16 transgenic mouse (29, 30).We assessed the two vaccines in mice bearing subcutaneousTC-1 and SC1 tumors, orthotopic TC-1 tumors at the cervico-vaginal tract (25), and experimental TC-1 lung metastases(31). The results presented below demonstrate that conjugat-ing an E7 SLP, which is currently being evaluated in clinicaltrials (7–10, 32), to a solid-phase platform has clear benefitscompared with a conventional free/liquid vaccine.

Materials and MethodsImmunization

Mice were immunized with a total amount of 15 mg of E7LP asfree peptide or in the NP-bound formulation, and 20 mg (sub-cutaneous TC-1 tumors experiment) or 40 mg (intravaginaltumors, lung metastases and SC1 experiments) of CpG was usedas adjuvant. For the free E7LP vaccine, the long peptide was firstdissolved in DMSO and then diluted in PBS prior to immuniza-tion. Control mice were treated with PBS. Groups were assignedby randomizing themice according to the tumor volume or to thebioluminescence signal to ensure a similar size distribution. Allthe mice from one experiment were immunized together on thesame day. All tumor-bearing mice were immunized once, unlessotherwise stated. Subcutaneous immunizations of mice wereperformed in the four limbs using the Hock method (33). Sub-cutaneous TC-1 tumor–bearing mice were immunized whenmean tumor size was around 100 mm3 (usually at day 10–12after implantation) unless otherwise stated, or when tumor sizewas around 170 mm3 for the anti-4-1BB combination experi-ment. Cervico-vaginal tumor-bearing mice were immunized sub-cutaneously in the back near the tail. Subcutaneous SC1 tumor–bearing FVB/N H2b mice were immunized once, 47 days after

tumor implantation when the mean tumor size reached approx-imately 100 mm3.

Mice, tumor cells, and antibody treatmentsFor all TC-1 implantation experiments, C57BL/6NCrl mice

were used. C57BL/6NCrl mice (aged 6–8 weeks) were pur-chased from Charles River and kept under pathogen-free con-ditions at the animal facility of Ecole Polytechnique F�ed�erale deLausanne (or at the animal facility of the Centre HospitalierUniversitaire Vaudois for the cervico-vaginal experiment). Allexperiments were performed in accordance with Swiss law andwith approval of the Cantonal Veterinary Office of Canton deVaud, Switzerland.

For the subcutaneous model, TC-1 cells (kindly provided byProf TC Wu, Johns Hopkins University, Baltimore, MD) werecultured in DMEM (Gibco), 10% FBS (Gibco), penicillin/strep-tomycin (100 U/mL penicillin, 100 mg/mL streptomycin; Gibco),andwere implanted subcutaneously on the flank of C57BL/6NCrlmice with inoculations of 5� 105 cells in 100 mL HBBS. The TC1cell line was authenticated by the expression of E6 and E7,cultured for 2 weeks (4–5 passages) prior implantation and testedforMycoplasma. Tumor growth was measured with a caliper usingthe formulaV¼W2Lp/6 andmicewere sacrificedwhen tumor sizereached 1,000 mm3.

For the cervico-vaginal tumor model, tumors were induced asdescribed in ref. 25. Briefly, anesthetized diestrus-synchronizedfemalemice were pretreated with 4% nonoxynol-9 (Abcam) for 6hours, and were then intravaginally implanted with 5� 104 TC-1cells engineered to express a luciferase reporter gene (TC-1-Luc).Tumor growth was monitored by bioluminescence index (BLI)using the Xenogen imaging system after intraperitoneal injectionof 50mg/kg D-luciferin (PerkinElmer) in PBS.Micewere sacrificedwhen interruption criteria defined in the animal license werereached.

For the lung metastasis model, 5 � 105 TC-1-Luc cells wereresuspended in 100 mL of PBS and injected into the tail vein offemale mice. Lung metastases were monitored by BLI afterintraperitoneal injection of 50 mg/kg D-luciferin (PerkinElmer)in PBS. Images were acquired using a Photon Imager (IVISSpectrum) system and data analyzed with the provided soft-ware (IVIS). Mice were imaged twice a week, and were sacrificedwhen interruption criteria defined in the animal license werereached.

SC1 cellswere generated froma squamous cell carcinomaof theskin of a K14HPV16/H-2b mouse (29). The K14HPV16/H-2b linewas generated by crossing K14HPV16/FVB/N (H-2q) mice(34–36) with C57BL/6 (H-2b) mice to introduce the H-2b locus.F1 mice were backcrossed for 11 generations to FVB/N, selectingfor the H-2b locus in every generation. This genetic configurationallows K14HPV16/H-2b mice to present E7-derived peptides onMHC-I molecules while maintaining the FVB/N background thatis permissive for squamous carcinogenesis. SC1 cells were cul-tured in DMEM (Gibco), 10% FBS (Gibco), penicillin/strepto-mycin (100 U/mL penicillin, 100 mg/mL streptomycin; Gibco),and 1 � 106 SC1 cells resuspended in 100 mL HBBS/Matrigel(Corning; 1:1 solution) were implanted subcutaneously on theflank of FVB/N H-2b mice. Tumor growth was measured with acaliper using the formula V ¼ W2Lp/6.

For CD4þ and CD8þ T-cell depletion, TC-1 tumor–bearingmice received intraperitoneal injections every 4 days of 10 mg/kgCD4 (clone GK1.5, BioXcell) and/or 10 mg/kg CD8 antibody

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(clone 53-6.7, BioXcell). Antibody treatment started 3 days beforevaccination and was continued for 3 weeks. CD4þ and CD8þ

T-cell depletionwasmonitoredweekly byflowcytometry analyseson the blood. Mice with less than 95% depletion were excludedfrom the analyses.

For the anti-4-1BB treatment, mice received 3 doses of 350 mgof anti-4-1BB (clone LOB12.3, BioXcell) intraperitoneally every3 days starting at day 12 together with the vaccine.

ReagentsCpG-B 1826 oligonucleotide (50-TCCATGAGCTTCCT-

GACGTT-30 as phosphorothioated DNA bases) was purchasedfrom Microsynth and used as an adjuvant in both vaccineformulations. HPV16 E7 long peptide GQAEPDRAHY-NIVTFCCKCDSTLRLCVQSTHVDIR (aa 43–77, purity > 90%)was purchased from Think Peptides and the Protein and Pep-tide Chemistry Facility, UNIL and used for NP conjugation andimmunization. HPV16 E7 CD8þ T-cell peptide RAHYNIVTFwas purchased from Think Peptides and used for restimulation.

NP synthesis and conjugationNPs were synthesized, functionalized, and characterized as

described previously (22, 23, 37). For antigen conjugation,HPV16 E7 long peptide was dissolved in DMSO and incubatedfor 12 hours in endotoxin-free water in the presence of NPs andguanidine hydrochloride (AppliChem) at room temperature. NP-E7LPwas purified by size-exclusion chromatography using CL-6Bmatrix (Sigma-Aldrich), eluted, and stored in PBS at room tem-perature. The size of NPs before and after conjugation wasdetermined by dynamic light scattering and remained around30 nm. E7LP loading on the nanoparticles was measured by BCAassay (Thermo Fisher Scientific). NPs alone have been shown tohave no adjuvant activity (38, 39).

Cell preparation for flow cytometry and antigen-specific in vitrorestimulation

Spleens were harvested and gently disrupted through a 40-mmfilter (Thermo Fisher Scientific). Red blood cells were lysed usingACK lysis buffer, and cells were filtered again through a 40-mmfilter before use. Tumors were harvested and minced usinga scalpel and digested for 45 minutes using collagenase A(0.33 U/mL, Roche), dispase (0.85 U/mL, Roche), DNAse I(144 U/mL, Roche) in RPMI medium with intermittent shakingat 37�C. For CD8þ T-cell antigen-specific restimulation, cellsuspensions from either spleen or tumor were cultured at 37�Cfor 6 hours in a 96 well plate in the presence of 1 mg/mL of theHPV16 CD8þ T-cell peptide RAHYNIVTF. After the first 3 hoursof culture, Brefeldin A (Sigma-Aldrich) was added to a finalconcentration of 5 mg/mL. All the cells were cultured in Iscove'smodified Dulbecco's medium (Gibco) supplemented with 10%FBS (Gibco) and penicillin/streptomycin (100 U/mL penicillin,100 mg/mL streptomycin; Gibco).

Flow cytometryFlow cytometry analyses were performed 9 days after immu-

nization of tumor-bearing mice. For surface staining and block-ing, cells were incubated for 15minutes on icewith the antibodiesdiluted in PBS 2% FBS. Before tetramer and antibody staining, allcell suspensions from tumor or spleen were blocked with antiCD16/32 (BioLegend). Cells were then labeled with fixable live/dead cell viability reagent (Invitrogen) in PBS for 15 minutes on

ice. E7-specific CD8 T cells were stained with a tetramer-recog-nizing HPV16 E7 peptide RAHYNIVTF (49-57) presented by H-2Db (University of Lausanne, UNIL, Lausanne, Switzerland) for30minutes at room temperature before surface antibody staining.If no intracellular staining was performed after surface staining,cells were fixed with 2% PFA in PBS for 15 minutes on ice. Forintracellular staining, cells were permeabilized and fixed with theFoxp3/Transcription Factor Staining Buffer Set Kit (eBioscience)following the manufacturer's instructions and then incubatedovernight with the antibodies diluted in 1� Permeabilizationbuffer provided with the kit. After staining, cells were washed andresuspended in PBS 2% FBS for analyses. Samples were acquiredon a Gallios analyzer (Beckman Coulter) and data were analyzedusing FlowJo software (Tree Star Inc.). For flow cytometry anal-yses, all lymphocytes were gated on single, live cells. Antibodiesused for flow cytometry: CD3 (clone 145-2C11, Thermo FisherScientific), CD4 (clone RM4-5, BioLegend), CD8a (clone 5H10,Thermo Fisher Scientific), B220 (clone RA3-6B2, Thermo FisherScientific), IFNg (clone XMG1.2, BioLegend), TNFa (clone MP6-XT22, Thermo Fisher Scientific), GZB (clone NGZB, ThermoFisher Scientific), Foxp3 (clone FJK-16s, ThermoFisher Scientific),CD25 (clone PC61.5, Thermo Fisher Scientific), CD45 (30-F11,Thermo Fisher Scientific), CD11b (clone M1/70, Thermo FisherScientific), CD11c (clone N418, BioLegend), Ly6G (clone 1A8,BioLegend), Ly6C (clone HK1.4, Thermo Fisher Scientific),CD206 (clone C068C2, BioLegend), MHCII (clone M5/114.15.2, BioLegend), 4-1BB (clone 17B5, BioLegend), GITR(clone DTA-1, BD Horizon), ICOS (clone 7E.17G9, BD Pharmin-gen), and OX-40 (clone OX-86, BioLegend).

Immunofluorescence stainingTumors were harvested, embedded in optimal cutting temper-

aturemedium (OCT; Sakura), and immediately frozen on dry ice.Ten-micron–thick sections were cut fromOCT-embedded tumorsusing a cryostat and collected on Superfrost Plus glass slides(Thermo Fisher Scientific). Tissue sections and OCT-embeddedtumors were stored at �80�C. For immunofluorescence staining,sections were fixed in ice-cold methanol (Thermo Fisher Scien-tific) for 10 minutes before proceeding. Slides were washed withPBS to remove the remaining OCT and then blocked for 45minutes at room temperature with PBS þ 5% BSA þ 2.5% FBSand then stained with primary antibodies diluted in PBS þ 1%BSA overnight at 4�C in a humidified chamber. On the followingday, slides were washed with PBS and stained with secondaryantibodies diluted in PBS þ 1% BSA for 1 hour at room temper-ature. Beforemounting, slides were washed again in PBS and thencovered with mounting media (Dako) containing DAPI (Roche,5 mg/mL). Coverslips (Menzel Glaser) were applied to the slidesand sealed using nail polish. Images were acquired using a LeicaDM5500B and processed using Fiji (ImageJ). The following anti-bodies were used for immunofluorescence staining: CD8 (clone53-6.7, eBioscience), keratin 14 (clone poly19053, BioLegend),ICOS (clone 7E.17G9, BD Biosciences), F4/80-PE (cloneBM8, eBioscience), CD11c-FITC (clone N418, BioLegend),MRC1-Alexa Fluor 647 (clone C068C2, BioLegend), andPD-L1-PE (MIH5, eBioscience).

RNA extraction and real-time PCRRNA from tumor tissue was isolated with the miRNeasy kit

(Qiagen) and 1 mg of total RNA was subjected to cDNA synthesisusing the iScript cDNA synthesis kit (Bio-Rad). Real-time PCRwas

Nanoparticle Conjugation Enhances HPV16 E7-SLP Vaccine

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conducted using the Rotor Gene SYBR green PCR kit (Qiagen)with 25 ng of cDNA on a Rotor Gene Q instrument (Qiagen) andthe following primers (Microsynth):

E7-fwd: CAGCTCAGAGGAGGAGGATG,E7-rev: GCCCATTAACAGGTCTTCCA,H2Db-fwd: AGTGGTGCTGCAGAGCATTACAA,H2Db-rev: GGTGACTTCACCTTTAGATCTGGG,B2M-fwd: TTCTGGTGCTTGTCTCACTGA,B2M-rev: CAGTATGTTCGGCTTCCCATTC,TAP1-fwd: GGACTTGCCTTGTTCCGAGAG,TAP1-rev: GCTGCCACATAACTGATAGCGA,Psmb5-fwd: GAGCCGCGAATCGAAATGC,Psmb5-rev: ATCCGCTGCAACAATGACTCC,RPL13a-fwd: CTGTGAAGGCATCAACATTTCTG,RPL13a-rev: GACCACCATCCGCTTTTTCTT.The relative amount of cDNA was calculated with the DDCt

method using RPL13a as the reference gene and the TC-1 tumorsample as calibrator.

DNA sequencingcDNA from TC-1 tumors was amplified with the E7-specific

primers ATGCATGGAGATACACCTAC and ATTATGGTTTCTGA-GAACAGA by Platinum Taq polymerase (Invitrogen) and clonedinto pSC-A employing the StrataClone PCR cloning kit (AgilentTechnologies). The insert containing theHPV16E7 gene sequencewas evaluated by Sanger sequencing using the T3 primer:TTAACCCTCACTAAAGG (Microsynth).

Statistical analysesStatistical analyses were performed in GraphPad Prism 7.

Flow cytometry data and CD8þ T cells quantification by histologywere compared using Student t test. Survival curves were com-pared using Log-rank (Mantel–Cox) test. Normalized growthcurves of SC1 tumors and mean TC-1 tumor growth curveswere compared using two-way ANOVA. Statistic significance isindicated as follows: �,P<0.05; ��,P<0.01; ��,P<0.001; ��,P<0.0001; n.s., not significant.

ResultsImmunization with NP-E7LP enhances systemic immuneresponses and prolongs survival

Classical (free-peptide/liquid) therapeutic vaccine formulationsagainst HPV16-driven cervical cancers have had equivocal thera-peutic responses against established carcinomas,motivating effortsto improve immunization strategies. Thus, we sought to determinewhether therapeutic vaccination with the NP-conjugated E7LP(NP-E7LP) could bemore effective than the free E7LP formulationat eliciting systemic E7-specific CD8þ T-cell responses inmice withsolid tumors, and to comparatively assess effects of the twovaccines on overall survival. C57Bl/6 mice were subcutaneouslyimplanted with TC-1 cells. When the mean size of the palpablesolid tumors was approximately 100mm3, we administered eitherNP-E7LPþCpG, free E7LPþCpG, or PBS as control (Fig. 1A).

We began by comparatively evaluating the systemic adaptiveimmune response. Tumor-bearing mice were sacrificed 9 days

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Figure 1.

NP-E7LP immunization increases the abundance of systemic E7-specific CD8þ T cells producing activation-associated cytokines, and improves survival oftumor-bearing mice. A, Schematic of the experimental layout. B, Flow cytometry analyses of E7-specific CD8þ T cells in the spleen. C, Representativeflow cytometry plots of tetramer staining for E7-specific CD8þ T cells gated on single cell live B220�CD3þCD8þ lymphocytes.D–G, Flow cytometry analyses of IFNg ,TNFa, and GZB production by CD8þ T cells after in vitro restimulation with the HPV16 E7 CD8þ T-cell peptide RAHYNIVTF. H, Survival of subcutaneous TC-1tumor-bearing mice. I, Individual TC-1 tumor growth curves. Groups: PBS (B–G, n ¼ 8; H and I, n ¼ 13), free E7LP (B–G, n ¼ 8; H and I, n ¼ 9), NP-E7LP(B–G, n ¼ 8; H and I, n ¼ 9), and NP-E7LP 2� (two inoculations of NP-E7LP; n ¼ 9). � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.

Galliverti et al.





after immunization and E7-specific CD8þ T cells in the spleenwere quantitated by flow cytometry analyses (Fig. 1B and C).Mice that received the NP-E7LP showed an increase in thefrequency of E7-specific CD8þ T cells that was 3- to 16-foldhigher compared with mice that were immunized with the freeE7LP. E7-specific CD8þ T cells were undetectable in PBS-treatedmice, suggesting the absence of a spontaneous CTL immuneresponse (Fig. 1B and C).

To comparatively evaluate the functionality of the E7-specificCD8þ T cells elicited by the two vaccine formulations, splenocytesharvested 9 days after immunization were restimulated in vitrowith the class I MHC–restricted E7 peptide RAHYNIVTF (26).NP-E7LP–treated mice showed a significant increase in cellsexpressing IFNg (Fig. 1D; Supplementary Fig. S1), TNFa(Fig. 1E; Supplementary Fig. S1), and Granzyme B (GZB;Fig. 1F; Supplementary Fig. S1) compared with mice that wereimmunized with free E7LP. A 3-fold increase in TNFa and IFNgdouble-positive cells was also detected in the NP-E7LP immu-nized mice, indicating that polyfunctional CD8þ T cells werebeing generated in higher proportions (Fig. 1G; SupplementaryFig. S1). Consistent with the observed absence of E7-specificCD8þ T cells in PBS-treated animals (Fig. 1B andC), the cytokinesand GZB were not detected in this group (Fig. 1D–F; Supplemen-tary Fig. S1). These results demonstrate that the NP-E7LP vaccineformulation is superior to the free E7LP vaccine at inducing asystemic CD8þ T-cell response, which is characterized by a largerpool of E7-specific cells and increased production of the proin-flammatory cytokines and GZB involved in killing target cells.

Congruent with the enhanced immune response elicited bythe NP vaccine, mice that received the NP-E7LP formulationsurvived significantly longer than those who received thefree form of E7LP, while all the PBS-treated control micereached the defined endpoint (tumor volume > 1 cm3) startingat day 20 postimplantation (Fig. 1H). Although overall survivalwas improved in both vaccinated groups, 5 of 9 tumors inmice treated with free E7LP did not respond to vaccination andkept growing, and these mice had to be euthanized around day20, similarly to PBS-treated mice (Fig. 1H and I). In contrast, 9of 9 NP-E7LP–treated mice showed tumor regression afterimmunization; moreover, in the NP-E7LP–treated group,tumor shrinkage was faster and tumor size remained stable(below 250 mm3) for a longer period than in the 4 of 9transitory responders to the free E7LP (Fig. 1I). Analysis of themean tumor volumes highlights the superior ability of NP-E7LP at causing tumor regression (Supplementary Fig. S2).Among the NP-E7LP–treated mice, 3 of 9 mice showed acomplete response with the disappearance of a palpable mass,and one was still tumor-free at day 137 postimplantation (Fig.1H and I). In contrast, none of the mice in the free E7LP–treatedgroup showed a complete response (Fig. 1I). Mice that receiveda second inoculation of NP-E7LP 7 days after the first one didnot show improvement in survival nor in tumor growth rate,compared with the group that received only a single dose of theNP-E7LP vaccine (Fig 1H and I; Supplementary Fig. S2).

To determine whether CD8þ and/or CD4þ T cells contribute tothe effects of NP vaccination, the NP-E7LP immunization of TC-1tumor–bearing mice was combined with CD4 or CD8 antibodytreatment (or a combination of the two). CD4þ T-cell depletiondid not affect the efficacy of the vaccine, revealing that CD4þ T-cellhelp is not required for the enhanced effect of the NP-E7LPformulation (Supplementary Fig. S3A–S3C). In contrast, CD8þ

T-cell depletion completely abolished the effect of the vaccine,demonstrating that the antitumor immune response elicited withNP-E7LP is dependent on CD8þ T cells (Supplementary Fig. S3A–S3C). Combined depletion of CD8þ andCD4þ T cells was similarto CD8þ T-cell depletion alone, supporting the conclusion thatCD4þ T cells do not play a role in the antitumor immune responsein this context (Supplementary Fig. S3A–S3C). Although the E7LPwe used contains both a CD4þ and a CD8þ T-cell epitope, in lightof the dispensability of the CD4þ T-cell response for the antitu-mor effects, we decided to focus subsequent experiments onCD8þ T cells.

Our initial experiments were designed to treat well-establishedsolid tumors. Next we vaccinated TC-1 tumor–bearing mice at anearlier timepoint, incipient neoplasia, similarly to what has beenreported in a previous study (40). We vaccinated at day 7 afterimplantation, when the mean tumor volume was approximately22 mm3, and 32% of the mice lacked a palpable mass. In thissetting, both formulations led to prolonged responses, althoughthe NP-E7LP vaccine appears superior (Supplementary Fig. S4AandS4B). As such, immunizationofmicewith incipient neoplasiamay not be fully informative about the effects of therapies aimedat well-established solid tumors, where the relative benefits of theNP-E7LP formulation are evident.

Tumors from NP-E7LP–treated mice have increased tumor-specific CD8þ T-cell infiltrates

We next assessed the abundance of E7-specific CD8þ T cellsinside the TC-1–derived tumors, and characterized associatedeffects on the tumor microenvironment (TME), comparing theNP-E7LP with the free E7LP vaccine. Using the previouslydescribed protocol (Fig 1A), TC-1 tumor–bearing mice weresacrificed 9 days after vaccination. Flow cytometry analyses ofthe tumors revealed a CD8þ T-cell response that reflected thesystemic responses described above in the spleen. Thus, tumorsharvested from mice treated with the NP-E7LP vaccine hadsignificantly higher CD8þ T-cell infiltrates compared with tumorsfrom free E7LP–treatedmice, while control mice treated with PBSdid not have appreciable CD8þ T-cell infiltration (Fig. 2A). Thefraction of E7-specific CD8þ T cells determined by tetramerstaining followed the same trend, with NP-E7LP–treated micehaving significantly more tumor-infiltrating antigen-specific cellsthan free E7LP–treated mice (Fig. 2B). Consistent with theseresults, immunostaining of tumor tissue sections from NP-E7LP–treated mice with anti-CD8 showed substantial infiltratesof CD8þ T cells distributed throughout the lesion. In contrast,tumors from the free E7LP–treated group containedonly scatteredCD8þ T-cell infiltrates, while CD8þ T cells were virtually absent inthe PBS-treated control groups (Fig. 2C; Supplementary Fig. S5).To assess the functionality of the tumor-infiltrating E7-specificcells, we performed a restimulation assay using disaggregated cellsfrom whole tumors. Intracellular staining for IFNg , TNFa, andGZB revealed that cytokine production by CD8þ T cells wassignificantly higher inmice that receivedNP-E7LP compared withfree E7LP, whereas it was virtually absent in tumors derived fromPBS-treated mice (Fig. 2D–F), underscoring the increasedimmune activity of the NP-E7LP vaccine over the free E7LPformulation.

We next analyzed innate immune cell infiltrates in treatedTC-1 tumors 9 days postvaccination. Irrespective of the formu-lation, vaccination caused a significant increase in intratumoralCD11bþF4/80þ macrophages compared with control PBS






treatment (Supplementary Fig. S6A). Despite the generalincrease, the subpopulation of M2-like CD206þ (MRC1þ)macrophages was significantly lower in both immunizedgroups (Supplementary Fig. S6B). Vaccination was also asso-ciated with an increase in CD11bHiLy6CþLy6G� monocytes(Supplementary Fig S6C); there was, however, no significantchange in CD11bHiLy6C�Ly6Gþ neutrophils, which were rarein infiltrating TC-1 tumors (Supplementary Fig. S6D). Althoughmore abundant in treated mice, no significant differences inF4/80�CD11bþCD11cþ and F4/80�CD11b�CD11cþ DCs weredetected between cohorts that received the two vaccines,although the F4/80�CD11b�CD11cþ DCs were significantlyincreased in the NP-E7LP–treated group compared with PBS-treated controls (Supplementary Fig. S6E and S6F). Thus, theNP-E7LP formulation behaved similarly to the free E7LP formin regard to eliciting innate immune infiltration, suggesting thatthe enhanced efficacy of the NP-E7LP vaccine is predominantlydue to a larger influx of tumor-specific and cytokine-producingCD8þ T cells rather than to specific changes in innate immunecell populations.

NP-E7LP limits intratumoral Treg accumulation and improvesCD8þ T-cell/Treg ratio

It has been shown that NPs can direct antigens toward theintracellular pathway that leads to peptide cross-presentation andloading onMHC-Imolecules (22, 23). Thus, we reasoned that the

NP E7LP-conjugated vaccine might be increasing peptide loadingonto MHC-I molecules and thereby impacting the intratumoralCD8þ T-cell/Treg ratio. We analyzed Treg cells infiltrating TC-1tumors by flow cytometry 9 days after immunization of tumor-bearingmice. In contrast to a previous study performedwith TC-1tumors, in which Tregs were reportedly abundant in untreatedtumors (41), we detected relatively few Tregs in this context.Vaccination with free E7LP led to a 4.5-fold higher proportionof intratumoral Tregs compared with the NP-E7LP–treatedgroup (Fig. 3A). Tregs were present in NP-E7LP–vaccinated miceat a similarly low frequency to the PBS-treated control mice(Fig. 3A). Consequently, the CD8þ T-cell to Treg ratio was con-siderably improved in NP-E7LP–treated tumors compared withthe free-E7LP group (Fig. 3B). Total CD4þ T-cell proportions wereunchanged upon vaccination with both formulations and similarto those in PBS-treated tumors (Fig. 3C). Thus, vaccination usingNP-bound antigen limited the generation and consequent intra-tumoral accumulation of Tregs following therapeutic vaccination.

NP conjugation improves long-peptide vaccination in differenttumor models

We next compared the NP-E7LP formulation with the conven-tional free long peptide vaccine against TC-1 tumors arising inthe cervico-vaginal tract and in lung metastases. To comparethe vaccines in an advanced disease setting, orthotopic cervico-vaginal TC-1 tumor–bearingmicewere immunized at day 14 after

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NP-E7LP immunization leads toincreased CD8þ T-cell infiltration andcytokine production in the tumormicroenvironment. Tumors wereanalyzed by flow cytometry (n¼ 8 pergroup) or embedded inOCT and frozenfor sectioning (n ¼ 4 per group).Flow cytometry analyses ofintratumoral CD8þ T cells (A) andE7-specific CD8þ T cells (B). C,Immunofluorescent staining for CD8(green) and nuclei (blue) on frozenOCT–embedded tumors, 4 samples pergroup were stained, analyzed, and arepresentative field of the tissue isshown. Scale bars, 100 mm. D–F, Flowcytometry analyses of IFNg , TNFa, andGZBproduction fromCD8þT cells afterin vitro restimulationwith the HPV16 E7CD8 peptide RAHYNIVTF. Groups:PBS (n ¼ 12), free E7LP (n ¼ 12),NP-E7LP (n ¼ 12). � , P < 0.05;�� , P < 0.01; �� , P < 0.001.

Galliverti et al.





implantation, when tumors were well established. NP-E7LP vac-cination significantly improved survival of tumor-bearing micewith25%survivingmore than150days; in the free E7LP group, allthe mice reached the endpoint shortly after 50 days (Fig. 4A andB). When mice bearing well-established TC-1 lung metastases (atday 9 post intravenous inoculation) were treated, (Fig. 4C andD),efficacy was also improved, with 28.5% of the mice surviving formore than 100 days postimplantation, whereas free E7LP vaccinetreatedmice all reached endstage before day 50. Tumors and lungmetastases collected fromNP-E7LP–immunizedmice 9 days aftervaccination showed an increase in CD8þ T-cell infiltrates com-pared with the free E7LP group, while PBS-treated control hadminimal CD8þ T-cell infiltrates in either setting (Fig. 4E and F;Supplementary Fig. S7A and S7B).

To test the efficacy of NP-E7LP immunization in anothersetting, we employed squamous carcinoma cells (SC1) thatexpress the HPV16 early region genes, representing the bona fidecell type transformed by HPVs. SC1 cells were implanted subcu-taneously, and mice were immunized whenmean tumor volumewas 100 mm3. Vaccination with NP-bound E7LP led to tumorregression/stabilization, whereas tumors from mice that receivedeither free E7LP or PBS kept growing (Fig. 4G). Treatment withNP-E7LP led to a significant increase in intratumoral CD8þ T cellscompared with the other groups (Fig. 4H and I). These assaysunderscore the superior efficacy of NP-E7LP over the free E7LPvaccine in physiologically distinct disease settings.

Efficacy-associated changes in the TME are progressively lostupon relapse

Despite increased systemic and local immune responses withthe NP-E7LP, most of the subcutaneous TC-1 tumors eventuallyrelapsed to progressive tumor growth after an initial phase of

tumor shrinkage (response) that was followed by an intermediatephase where the tumor size remained relatively stable for a periodranging from10 to 50 days (Fig. 1I).We sought to characterize thechanges in the TME that are associated with these three phases(Supplementary Fig. S8A) in mice that received the NP-E7LPvaccine.

We analyzed expression of the E7 oncogene, potential muta-tion of the E7-encoded CD8þ T-cell peptide epitope containedwithin the SLP vaccine, and expression of several components ofthe antigen processing and presentation machinery, comparingresponding tumors with relapsed ones. We found no significantchange that could explain the loss of responsiveness toward thecancer cells (Supplementary Fig. S8B).

We then examined the T cells in the TME and found that CD8þ

T-cell infiltration progressively decreased after the response phase(Fig. 5; Supplementary Fig. S9A). CD8þ T-cell activation, asindicated by ICOS staining, was high in the response phase butwas already lost in the stable disease phase (Fig. 5; SupplementaryFig. S9B).

Macrophageswere present in the TME at all stages, although theexpression of F4/80 appeared to be upregulated in respondingand stable tumors (Fig. 5, Supplementary Figs. S9C and S10). TheDC- and M1-macrophage marker CD11c (42) was absent in PBS-treated tumor sections but present throughout the sections ofresponse-phase tumors, and then progressively lost during tran-sition to stable disease and relapse phase tumors (Fig. 5; Supple-mentary Figs. S10 and S11A). Staining for the M2 macrophagemarker MRC1 (CD206; ref. 42) was conversely increased inuntreated and relapsed tumors (Fig. 5; Supplementary Figs. S10and S11B). These data indicate that the abundance of DCs and thefrequency and phenotype of macrophages changes concordantwith CD8þ T-cell activation. It has previously been reported that

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NP conjugation of E7LP preventsthe accumulation of intratumoralregulatory T cells upon vaccination.A, Flow cytometry analyses ofCD4þFoxp3þCD25þ regulatory T cells inthe tumor. B, The ratio of CD8þ T cellsto CD4þFoxp3þCD25þ regulatory T cellsin TC-1 tumors calculated from flowcytometry data. C, Flow cytometryanalyses of total CD4þ T cells in thetumor. Groups: PBS (n ¼ 7), free E7LP(n ¼ 7), NP-E7LP (n ¼ 7). � , P < 0.05;�� , P < 0.001; n.s., not significant.






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NP conjugation improves survival and CD8þ T-cell infiltration in mice with cervico-vaginal tumors, or lung metastases, or squamous cell carcinomas. A, Survival ofcervico-vaginal TC-1 tumor-bearing mice. B, Representative bioluminescence images of cervico-vaginal TC-1 tumor-bearing mice at the indicated timepoints aftercell implantation, with day 14 representing the starting day of the treatment. C, Survival of TC-1 lung metastasis–bearing mice. D, Representative bioluminescenceimages of TC-1 lung metastases-bearing mice at the indicated timepoints after cell implantation, with day 9 representing the starting day of the treatment. E,Immunofluorescence staining for CD8 (green) and nuclei (blue) on frozen OCT-embedded cervico-vaginal tumors harvested 9 days after immunization, 3 (PBS andNP-E7LP) or 4 (free E7LP) samples were stained, analyzed, and a representative field of the tissue is shown. Scale bars, 100 mm. F, Immunofluorescencestaining for CD8 (green) and nuclei (blue) of lungmetastases derived from frozenOCT–embedded lungs harvested 9days after immunization, 4 (PBS) or 8 (free E7LPandNP-E7LP) sampleswere stained, analyzed, and a representative field of the tissue is shown. Scale bars, 100 mm.G, SC1 tumor growth normalized to the initial sizeat the time treatment commenced. Black stars refer to PBS versus NP-E7LP; green stars refer to free E7LP versus PBS; blue stars refer to NP-E7LP versus freeE7LP. H, Quantification of intratumoral CD8þ T cells performed on histologic sections. The values are calculated as the percentage of DAPIþ CD8þ T-cellnuclei area to the total DAPIþ nuclei area inside the keratin 14þ tumor area. I, Immunofluorescence staining for CD8 (green), keratin 14 (K14, red), and nuclei (blue) onfrozen OCT-embedded tumors. Scale bars, 100 mm. Cervico-vaginal TC-1 tumors groups: PBS (n ¼ 14 þ 3 for histology), free E7LP (n ¼ 16 þ 4 for histology),NP-E7LP (n¼ 12þ 3 for histology). TC-1 lungmetastases groups: PBS (n¼ 10þ 5 for histology), free E7LP (n¼ 7þ 8 for histology), NP-E7LP (n¼ 7þ 8 for histology).SC1 tumor–bearing mice groups: PBS (n ¼ 9), free E7LP (n ¼ 9), and NP-E7LP (n ¼ 9). � , P < 0.05; �� , P < 0.01; n.s., not significant.

Galliverti et al.





Figure 5.

Analyses of the tumor microenvironment at different stages of disease progression. Immunofluorescent staining for CD8, ICOS, F4/80, CD11c, MRC1, andPD-L1 on frozen OCT-embedded tumors collected at the indicated timepoints as in Supplementary Fig. S8A. PBS-treated tumors were collected at the sametime after TC-1 cell implantation as tumors in the response phase; at this timepoint, PBS-treated tumor were already at the endpoint. Scale bars, 100 mm.Groups: PBS (n ¼ 2), NP-E7LP response (n ¼ 4), NP-E7LP stable (n ¼ 4), and NP-E7LP relapse (n ¼ 4).






macrophages are modulated by the activity of CD8þ T cells andrequired for the antitumor response against TC-1 tumors elicitedby vaccination (24). As such, it seems likely that the DC andmacrophage dynamics in the stable disease and relapse phases areconsequent to reduced recruitment of activated CD8þ T cells.

Analysis of PD-L1 expression revealed that it was upregulated inthe response phase when activated CTLs are abundant, likelyinduced by CD8þ T-cell–secreted IFNg , whereas it was virtuallyabsent in untreated tumors and in the stable and relapse phases(Fig. 5; Supplementary Fig. S11C), suggesting that PD-L1 expres-sion is not the primarymechanism responsible for the progressiveloss of CD8þ T cells.

NP-E7LP synergizeswith agonistic anti-4-1BB/CD137 to furtherboost tumor rejection

Early immunization with a traditional free SLP vaccine of TC-1tumor–bearing mice led to discernibly enhanced immuneresponses and survival when given in combination with anagonistic anti-4-1BB antibody (40). In light of the evident lossof CD8þ T-cell activity in the stable disease and relapse phases(Fig. 5; Supplementary Fig. S9B), we comparatively evaluated theeffects of anti-4-1BB in the context of both vaccines. To extend theprevious study andmore rigorously evaluate the potential benefit

of this agonistic antibody, we treated larger, later-stage tumorsthat are poorly responsive to immunization with free E7LP alone.TC-1 tumor–bearing mice were immunized when mean tumorvolume was around 170 mm3. Mice treated with free E7LPreached endstage within 3 weeks after implantation of TC-1cells, whereas, as in previous experiments, all the mice treatedwith the NP-E7LP formulation survived significantly longer (Fig.6A and B). Anti-4-1BB significantly improved survival in combi-nation with both vaccine formulations (Fig. 6A and B). Theimproved efficacy of free E7LP þ anti-4-1BB was comparablewith that of NP-E7LP alone, and the inclusion of anti-4-1BBfurther improved its therapeutic efficacy. Comparison of meantumor volumes in the treatment cohorts (Supplementary Fig.S12) was time-limited and thus not fully informative due to thestatistical necessity of prematurely terminating the comparisonwhen the first free E7LP–treated mice reached the veterinary-defined endpoints.

To investigate the mechanism by which anti-4-1BB increasedthe efficacy of the two vaccines, we analyzed intratumoralimmune infiltrates 9 days postimmunization with and withoutanti-4-1BB treatment. The anti-4-1BB treated groups showed onlya modest increase in total E7-specific CD8þ T-cell infiltrates(Supplementary Fig. S13A). However, we observed an increase

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Therapeutic vaccination in combinationwith the agonistic antibody anti-4-1BB.A, Survival of subcutaneous TC-1 tumor–bearing mice; NP-E7LP versus freeE7LP P ¼ 0.0007, NP-E7LP versus freeE7LP þ anti-4-1BB P ¼ 0.0855, NP-E7LPversus NP-E7LPþ anti-4-1BB P¼ 0.0001,NP-E7LPþ anti-4-1BB versus free E7LPþanti-4-1BB P ¼ 0.0260, free E7LP versusfree E7LP þ anti-4-1BB P ¼ 0.0016. B,Individual TC-1 tumor growth curves.Groups: free E7LP (n ¼ 7), NP-E7LP(n ¼ 8), free E7LP þ anti-4-1BB (n ¼ 7),and NP-E7LP þ anti-4-1BB (n ¼ 8). �, P <0.05; �� , P < 0.001; n.s., not significant.

Galliverti et al.





in CD44þKLRG1þ terminal effector E7-specific CD8þ T cells(Supplementary Fig. S13B) in the context of anti-4-1BB treatmentthat correlated with a significantly higher infiltration of these cellsinto the tumors, particularly in the cohort treated with NP-E7LPvaccine plus anti-4-1BB (Supplementary Fig. S13C). The CD8þ

T-cell/Treg cell ratio showed a trend toward an increase upon anti-4-1BB treatment in the NP-E7LP group (Supplementary Fig.S13D). We also observed a trend toward increased frequency ofE7-specific CD8þ T cells expressing activation markers (4-1BB,GITR, ICOS, and OX-40; Supplementary Fig. S13E–S13H) andelevated percentages of IFNg-, TNFa-, and GZB-producing E7-specific CD8þ T cells upon anti-4-1BB treatment (SupplementaryFig. S13I–S13K). These data underscore the enhanced therapeuticefficacy of the NP vaccine in combination with anti-4-1BB.

DiscussionTherapeutic vaccination is an attractive strategy to direct the

immune system against specific cancer-associated antigens insolid tumors. CD8þ T cells play a fundamental role in tumorrejection, and thus induction of a potent CD8þ T-cell response is amajor goal of therapeutic vaccines. Cervical cancer andotherHPV-induced malignancies may be attractive prototypes for immuno-therapy in general and therapeutic vaccines in particular, giventhat the viral oncogenes encode neoantigenic oncoproteins thatdrive the disease. In patients with HPVþ tumors, generatingtherapeutically efficacious CTL responses has proven difficult toachieve with classical vaccination strategies involving peptide/protein vaccines (43), including studies utilizing the HPV E7-derived SLP that produced, at best, modest CD8þ T-cell responses(7–9, 14). In terms of efficacy, E7 SLPs have proven to be effectivein a minority of patients bearing early premalignant HPVþ

lesions, whereas bona fide malignancies have been largely refrac-tory (7, 10), underscoring the need for better therapeuticmodalities.

Herein we show that by conjugating an E7-derived SLP tosynthetic ultra-small (30 nm) NPs, it is possible to significantlyboost the CD8þ T cell response following immunization (by 3–16 fold), consistent with the previously reported (18, 22, 23)ability of NPs to enhance MHC-I antigen presentation byefficient delivery of their antigenic payload to cross-presentingDCs. The NP-E7LP vaccine formulation was capable, in com-parison with the non-NP–conjugated free E7LP, of eliciting asystemic immune response characterized by a larger pool of E7-specific CD8þ T cells producing activation-associated cytokinesand GZB.

The NP-E7LP vaccine induced greater infiltration of tumors byCD8þ T cells in both subcutaneous and cervico-vaginal TC-1tumors aswell as in TC-1–derived lungmetastases, in comparisonwith the free E7LP. The results highlight the ability of theNP-E7LP–generated CD8þ T cells to efficiently reach differentanatomic locations and perform their effector function. Thera-peutic vaccination with NP-E7LP of mice bearing TC-1–derivedsolid tumors led to tumor shrinkage in 100% of the treatedanimals, significantly increasing their lifespan. In contrast, in thisdisease setting, free E7LP only produced a response in about 50%of the mice. The NP-E7LP vaccine conveyed long-term survival in25% and 28.5% of the mice with orthotopic TC-1 tumors orexperimental lung metastases, respectively, whereas all of micetreated with the free E7 long peptide rapidly succumbed totheir disease. Similar results were observed in mice bearing

subcutaneous HPV16þ squamous cell carcinomas, extending thecomparative benefits to the histologic tumor type associated withHPV transformation. Thus, in SC1 tumor–bearingmice, NP-E7LPimmunization led to higher intratumoral CD8þ T-cell infiltratescompared with the classical free E7LP, resulting in tumor regres-sion/stabilization in marked contrast to the free E7LP vaccine,which was ineffective this setting.

These data clearly demonstrate that NP conjugation canimprove upon the capability of SLPs to induce tumor antigen–specific CD8þ T cells in sufficient quantity and activity to producesubstantive regressions of well-established solid tumors, leadingto appreciable survival benefit.

Promising results have also been reported for other distinctvaccine delivery platforms based on larger particles (44, 45) andlymph node–targeting vaccines, either alone (19) or in complexcombinatorial strategies (46). Compared with an impressive butelaborate combinatorial therapeutic regimen (46), we show that arelatively simple approach, solely based on conjugation of anantigenic SLP to a NP-based delivery system and its administra-tion in a single dose is sufficient to elicit a significant antitumorresponse not achievable with a traditional unconjugated "liquid"formulation.

Collectively, the results from the current study and otherreports clearly demonstrate that a variety of solid-phase andlymph-node targeting systems have considerable potential toimprove upon the generation of immune responses followingtherapeutic vaccination. It will be auspicious in future studiesto compare these distinctive antigen delivery systems head-to-head in the same experimental setting, to rigorously assess theirrelative benefits.

We have, in addition, confirmed the results of a previousstudy describing the potential of combining therapeutic vacci-nation with anti-4-1BB (40), strengthening the proof of conceptby demonstrating herein that this agonistic antibody can bebeneficially combined with a lymph node targeting solid-phaseantigen delivery system. Collectively, the data establish thatanti-4-1BB treatment promotes the generation of terminaleffector CD8þ T cells that infiltrate the tumors in highernumbers, are more activated, and produce higher amounts ofcytokines. We suggest that combinations of novel antigen-delivery platforms, such as the NPs described herein, withagonistic antibodies, including anti-4-1BB/CD137, could pres-ent a new foundation upon which to build future treatmentstrategies for HPVþ cancer patients.

Another recognized limitation of therapeutic vaccines based onSLPs is the concomitant generation of Tregs in response to theimmunization (16, 40), an undesirable response that has alsobeen observed in HPVþ cancer patients, particularly those withlarger/advanced lesions (7). Consistent with this result, weobserved that immunization of tumor-bearing mice with freeE7LP was associated with an increase in tumor-infiltrating Tregsthat resulted in a low CD8þ T-cell/Treg ratio. In contrast, immu-nization with the NP-E7LP did not increase intratumoral Treginfiltrates, resulting in a much higher CD8þ T-cell/Treg ratio.These data suggest that, by means of NP conjugation it may bepossible to restrict the generation of Tregs and thereby helpcircumvent one of the limitations of SLP vaccines. This effect islikely due to the ability of NPs to direct the delivery of antigens tothe intracellular processing pathway responsible for peptide cross-presentation on MHC class I molecules (22, 23) ultimatelyfavoring a higher intratumoral CD8þ T-cell/Treg ratio.






Despite the substantial increase in both systemic and localantitumor immune responses achievable with the NP-E7LP vac-cine, we observed that most tumors eventually relapsed. Tumorshrinkage was followed by a relatively long stable-disease phasethat lasted up to 50 days in subcutaneously transplanted mice,followed by relapse to progressive tumor growth. Our dataindicate that the relapse is not due to a loss of antigen presentationby tumor cells, but it is associatedwith a reduction of intratumoralCD8þ T cells and possibly a loss of their activity. These changes areevidently not caused by the presence of immunosuppressive M2macrophages or by expression of the inhibitory molecule PD-L1(47, 48). We envisage that the treatment regimen with NP-boundSLP described herein could be used as a model in which to studychanges associated with immunoediting (49) as well as strategiesaimed to reactivate the immune response during the stabledisease/equilibrium phase, which could be relevant for patientswith cancer treated with immunotherapies. This escape mecha-nism could explain why, in subcutaneous TC-1 tumor–bearingmice, a second inoculation of the NP-E7LP vaccine, performed 7days after the first one, failed both to promote greater tumorshrinkage and to improve survival, compared with mice thatreceived only a single administration.

In conclusion, our data illustrate the potential for this particularand for other NP-based lymphnode–targeting platforms (19, 44–46) to serve as effective delivery vehicles that boost the efficacy ofneoantigen-based therapeutic vaccines in patients, most obvious-ly to target HPVþ cancers with the well-validated E7LPs, butpotentially also other cancers for which stimulatory neoantigenshave been identified (50). It is possible that the use of antigenicpeptides coupled with such systems will enhance many immu-notherapeutic vaccine strategies for solid tumors, whether singleor combinatorial, involving SLPs. This proposition warrants con-sideration for FDA investigational new drug, enabling vaccinedevelopment and clinical evaluation.

Disclosure of Potential Conflicts of InterestM.A. Swartz has ownership interest (including stock, patents, etc.) in a patent

on the nanoparticle technology, but it is not licensed. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Galliverti, M. Tichet, D. Nardelli-Haefliger,M.A. Swartz, D. Hanahan, S. WullschlegerDevelopment of methodology: G. Galliverti, S. Hauert, M.A. SwartzAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Galliverti, M. Tichet, S. Domingos-Pereira,S. WullschlegerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Galliverti, M. Tichet, S. Domingos-Pereira,M.A. Swartz, D. Hanahan, S. WullschlegerWriting, review, and/or revision of the manuscript: G. Galliverti, M. Tichet, S.Domingos-Pereira, S. Hauert, D. Nardelli-Haefliger, M.A. Swartz, D. Hanahan,S. WullschlegerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Galliverti, S. WullschlegerStudy supervision: D. Nardelli-Haefliger, M.A. Swartz, D. Hanahan,S. Wullschleger

AcknowledgmentsWe thank B. Torchia, M.-W. Peng, and M.A. Gaveta for technical support;

Dr. R. Guiet and the Bioimaging and Optics Platform (BIOP) Core Facility atEPFL for developing image analyses tools; Prof. T.C. Wu from Johns HopkinsUniversity for providing TC-1 cells; and Prof. Daniel E. Speiser and Prof. PedroRomero for comments on the manuscript. This work was supported by aSinergia grant 160742 from the SNSF.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 15, 2018; revised June 16, 2018; accepted August 16, 2018;published first August 21, 2018.

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2018;6:1301-1313. Published OnlineFirst August 21, 2018.Cancer Immunol Res Gabriele Galliverti, Mélanie Tichet, Sonia Domingos-Pereira, et al. Tumors in MicePeptides Enhances Therapeutic Vaccine Efficacy against Solid Nanoparticle Conjugation of Human Papillomavirus 16 E7-long

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