cancer immunology research - efficient eradication of established tumors … · 2017. 1. 31. ·...

Efficient eradication of established tumors in mice with cationic

liposome-based synthetic long-peptide vaccines

Eleni Maria Varypataki a, Naomi Benne a, Joke Bouwstra a, Wim Jiskoot a,*, Ferry Ossendorp b,*

a Division of Drug Delivery Technology, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research,

Leiden, The Netherlands

b Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The

Netherlands

* Corresponding authors:

Ferry Ossendorp, Department of Immunohematology and Blood Transfusion, Leiden University

Medical Centre, P.O. Box 9600, 2300 RC Leiden, The Netherlands; Email:

[email protected]; Phone: +31 71 526 3843; Fax: +31 71 526 5257

Wim Jiskoot, Division of Drug Delivery Technology, Cluster BioTherapeutics, Leiden Academic Centre

for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands;

Email: [email protected]; Phone: +31 71 527 4314; Fax: +31 71 527 4565

Running title: Liposomal peptide vaccines for cancer immunotherapy

Funding

This study was financially supported by the funding organization, "Leidse profileringsgebied

Translational Drug Discovery and Development en de samenwerking LACDR en LUMC" and a CRI

Clinic and Laboratory Integration Program (CLIP) Grant (FO).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest are disclosed by the authors.

Keywords: cancer immunotherapy, cationic liposomes, synthetic long peptides, HPV E7, melanoma, TLR ligand

on July 22, 2021. © 2017 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 31, 2017; DOI: 10.1158/2326-6066.CIR-16-0283

http://cancerimmunolres.aacrjournals.org/

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A B S T R A C T

Therapeutic vaccination with synthetic long peptides (SLPs) can be clinically effective against HPV-

induced pre-malignant lesions; however, their efficiency in established malignant lesions leaves

room for improvement. Here, we report the high therapeutic potency of cationic liposomes loaded

with well-defined tumor-specific SLPs and a TLR3 ligand as adjuvant. The cationic particles, with an

average size of 160 nm, could strongly activate functional, antigen-specific, CD8+ and CD4+ T cells and

induced in vivo cytotoxicity against target cells after intradermal vaccination. At a low dose (1 nmol)

of SLP, our liposomal formulations significantly controlled tumor outgrowth in two independent

models (melanoma and HPV-induced tumors) and even cured 75%–100% of mice of their large

established tumors. Cured mice were fully protected from a second challenge with an otherwise

lethal dose of tumor cells, indicating the potential of liposomal SLP in the formulation of powerful

vaccines for cancer immunotherapy.




3

Introduction

Antigen-specific immunotherapy by vaccination is a promising approach for activation of tumor-

specific T cells in patients. Intensive research on the application of therapeutic vaccines for

treatment of numerous tumors, including melanoma, lung, and cervical cancers, is intensifying (1-4).

Preventive vaccines against infections with the high-risk types of HPV have been successfully

introduced to reduce the mortality associated with human papillomavirus (HPV)-induced cervical

cancer (5). However, these vaccines have little effect against established genital lesions, for which

improved cellular immunity is needed, including HPV-specific CD4+ T helper (TH) and CD8+ cytotoxic T

lymphocytes (CTL) (6, 7). CD8+ T cells are known for 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 the E6 and E7 oncogenic proteins as the most common targets for the

development of therapeutic vaccination against HPV-induced tumours (9, 10).

Immunotherapy using synthetic long peptides (SLP) of the HPV16 E7 oncoprotein efficiently

eradicates HPV16+ established tumors in mice (11). Patients with high-grade vulvar intraepithelial

neoplasia vaccinated with a mix of SLPs against E6 and E7 proteins have clinical responses, with 47%

of them showing complete regression of their pre-malignant lesions after one year (12). However,

this E6/E7 SLP-based vaccine is not effective in end-stage HPV16+ cervival cancer patients (2). The

SLP vaccines used so far for clinical trials have been emulsified in a clinical grade version of

incomplete Freund’s adjuvant, Montanide ISA-51, a suboptimal formulation adjuvant with reported

adverse effects such as local swelling, pain, redness, and in some cases fever (12).

As an alternative to Montanide, we propose liposomes: liposomes are promising as antigen-delivery

system in vivo, able to enhance antigen uptake by dendritic cells (DCs) and the subsequent initiation

of cell-mediated antitumor immune responses upon vaccination (13-16). Cationic liposomes are

considered to be more immunogenic than neutral or anionic ones (17, 18). We reported encouraging

preclinical results of intradermally administered cationic liposomal SLP formulations using model




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ovalbumin (OVA)-derived SLPs , showing potent in vivo T-cell priming capacity (19). The direct

comparison of these liposomes with PLGA nanoparticles, Montanide ISA-51, and a squalene-based

emulsion formulation revealed the superior capacity of these liposomes to raise effective cytotoxic

T-cell responses (20). This observation, together with the unfavorable safety profile of the currently

used adjuvant Montanide ISA-51, makes liposomes attractive candidates as delivery platforms for

SLP-based immunotherapy of cancer.

In this study, we analyzed the potency of our SLP-loaded DOTAP [1,2-dioleoyl-3-

(trimethyammonium) propane]–based cationic formulation as a therapeutic cancer vaccine in two

independent tumor models. The ovalbumin-derived SLPs containing CTL and TH epitopes (20) were

loaded into liposomes, combined with different TLR ligands (poly(I:C), Pam3CysK4, CpG) and the

most potent formulations were applied in a foreign antigen (OVA)-expressing melanoma model. In

an independent setting, HPV16 E7 SLP was formulated in the same liposomal system and analyzed as

a therapeutic vaccine in the TC-1 HPV+ tumor model. Both formulations were highly effective in the

induction of cellular immunity and tumor control.




5

Materials and Methods

Materials

The ovalbumin-derived SLP OVA24 [DEVSGLEQLESIINFEKLAAAAAK] and the short CTL-epitope

peptide OVA8 [SIINFEKL] were produced at the GMP facility of the Clinical Pharmacy and Toxicology

Department at the Leiden University Medical Center. The OVA17 SLP [ISQAVHAAHAEINEAGR],

including the OVA TH epitope AAHAEINEA and the 21-mer amino-acid long SLP,

GQAEPDRAHYNIVTFCCKSDS, harboring the CTL epitope RAHYNIVTF (H-2Db) (49-57) and the TH

epitope PDRAHYNIVTF (48-57) of HPV-16 E7 protein, were produced in the Immunohematology and

Blood Transfusion Department of the Leiden University Medical Center. The lipids DOPC [1,2-

Dioleoyl-sn-glycero-3-phosphocholine] and DOTAP were purchased from Avanti Polar Lipids

(Alabaster, Alabama, USA) and the TLR ligands (poly(I:C), Pam3CSK4, and CpG) with their labeled

analogs were obtained from InvivoGen (Toulouse, France). Carboxyfluorescein succinimidyl ester

(CFSE) was purchased from Invitrogen (Eugene, Oregon, USA). Acetonitrile (ACN), chloroform, and

methanol were obtained from Biosolve BV (Valkenswaard, the Netherlands) and Vivaspin 2

membrane concentrators were purchased from Sartorius Stedim Biotech GmbH (Goettingen,

Germany). Iscove’s modified Dulbecco’s medium (IMDM; Lonza Verniers, Belgium) was

supplemented with 8 % (v/v) fetal calf serum (Greiner Bioscience, Alphen a/d Rijn, the Netherlands),

50 µM 2-mercaptoethanol (Sigma-Aldrich, Zwijndrecht, Netherlands), penicillin (100 IU/mL) and 2

mM glutamine (Life Technologies, Bleiswijk, the Netherlands). MQ water and 10 mM PB (7.7 mM

Na2HPO4. 2 H2O and 2.3 mM NaH2PO4

. 2 H2O), were filtered through a 0.22-µm Millex GP PES-filter

(Millipore, Darmstadt- Germany) before use. Phosphate-buffered saline (PBS) was purchased from B.

Braun (Meslungen, Germany).

Mice




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Female C57BL/6 (H-2b) mice were purchased from Charles River (L’Arbresle, France) and congenic

CD45.1 (Ly5.1) mice were bred at the Leiden University Medical Center animal facility and used at 8-

14 weeks of age according to the Dutch Experiments on Animal Act, which serves the

implementation of “Guidelines on the protection of experimental animals” by the Council of Europe.

All performed animal experiments were approved by the animal experimental committee of Leiden

University.

Liposome preparation

Cationic liposomes of DOTAP and DOPC (molar ratio 1:1) loaded with OVA24 and OVA17 were

prepared by using the thin film dehydration-rehydration method, as described previously (19, 20).

For the liposomes loaded with E7 SLP the aqueous solution of the SLPs was first adjusted to a pH of

about 8.5. For poly(I:C) or CpG loaded liposomes, the ligand (including 0.5% fluorescently-labeled

equivalent) was added dropwise to the dispersion in a total concentration of 200 µg/mL. For

liposomes loaded with the lipophilic Pam3CysK4, the TLR ligand was dissolved in chloroform

together with the lipids, before the dry film formation.

Sizing of the liposomes and purification were performed as described previously (19, 20), followed

by the particles’ physicochemical characterization (average diameter (Zave), polydispersity index

(PDI), zeta-potential determination) as well as measurement of their SLP-content (UPLC) and the

amount of the loaded TLR ligand (fluorescence measurement in collected non-solubilized samples)

(19, 20).

Vaccination of mice

Mice were immunized by intradermal injection on the abdominal or tailbase skin area. All

formulations were prepared on the day of injection. Vaccination dose was based on the OVA24 or E7

SLP concentration, 1 nmol (2.5 µg for OVA24 or 2.3 µg for E7 SLP) of peptide in a total volume of 30

µl, and immunizations were performed on day 0 (prime immunization) and on day 14 (boost




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injection). Vaccinations with adjuvanted liposomes included a dose of 0.5 – 1.0 µg of TLR ligand.

During the in vivo studies, blood samples were obtained from the tail vein at different time points.

Analysis of antigen-specific CD8+ and CD4+ T-cell responses by flow cytometry

Staining of the cell surface was performed on blood samples and splenocytes after red blood cell

lysis. Cells were stained in staining buffer for 30 min with allophycocyanin (APC)-labeled H-2 Kb-

SIINFEKL tetramers or allophycocyanin (APC)-labeled H-2Db - RAHYNIVTF (E749-57) and fluorescently

labeled antibodies specific for mouse CD3 (BD Biosciences, Breda, the Netherlands), CD4 and CD8

(eBiosciences). 7-Aminoactinomycin D (7AAD) (Life Technologies) was used for the exclusion of dead

cells.

Overnight intracellular cytokine analysis of PBMCs was performed after incubating the cells with 2

µM of OVA8 and 2 µM of OVA17 or 2 µM of E7 SLP in presence of brefeldin A (7.5 µg/mL) (BD

Biosciences). The next day the assay was developed as described (19, 20).

In vivo cytotoxicity assay

Splenocytes from naive congenic CD45.1+ mice were labeled with CFSE for 1 h at 37oC with either 5

µM (target population) or 0.5 µM (control) final concentration and pulsed for 1 h with the short

SIINFEKL (OVA SLP) (19) or RAHYNIVTF (E7 SLP) peptide in complete culture medium at 37oC. Cells

were adoptively transferred intravenously in a 1:1 number ratio in recipient previously immunized

C57BL/6 mice and two days afterwards (day 24) they were sacrificed and single cell splenocyte

suspensions were analyzed for specific killing (SK) following the equation:

SK = 1 − CFSE highCFSE low vaccinated miceCFSE high CFSE low naive mice x 100 %

Tumor regression experiment




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B16-OVA melanoma or TC-1 tumor cells expressing HPV16-E7 proteins were cultured at 37oC with

5% CO2 in IMDM containing 8% fetal calf serum + 2 mM glutamine and 100 IU/mL penicillin in the

presence of 1 mg/mL for B16-OVA or 400 μg/mL geneticin (G-418) for TC-1 (Life Technologies), non-

essential amino acids (10X) (Life Technologies) and 1 mM sodium pyruvate (Life Technologies).

On day 0, mice were injected subcutaneously in the flank with 1x105 tumor cells in 100 μl PBS. On

day 9, when tumors were palpable (> 8 mm3) or day 12, when tumors had a large size (> 100 mm3)

mice were split into groups with similar tumor size and were vaccinated, as described above. Non-

immunized mice injected with tumor cells were used as a negative control.

The mice were weighed three times a week and their tumor size was measured in three dimensions

using a caliper. Mice having a tumor size > 2000 mm3 were sacrificed for ethical reasons.

In vivo CD8+ T-cell depletion

For the in vivo CD8+ T-cell depletion, the tumor-bearing mice were injected i.p. with 100 mg of the

monoclonal antibody (mAb), isolated from the 2.43 hybridoma cells that specifically recognizes CD8,

on days -1, 1, 7 and 14 after vaccination. The antibody was prepared and purified as described (21).

Statistical analysis

The significance of differences in the in vitro and in vivo assays were evaluated by GraphPad Prism 6

(GraphPad) software, by using an analysis of variance (ANOVA) at a 0.05 significance level, followed

by Bonferroni’s post-test. The significance of differences between the survival curves was calculated

with the log-rank (Mandel-Cox) test.




9

Results

Characterization of liposomes loaded with synthetic peptide antigens and adjuvant

Following the optimized protocol for the preparation of synthetic long peptide (SLP)–loaded

liposomes (20), liposomes loaded with OVA- or HPV E7-derived SLPs were produced. Entrapment of

SLPs in the liposomes appeared to be highly dependent on electrostatic interactions between the

liposomes and the peptides (19, 20) (Supplementary Table S1 and Supplementary Fig. S1). The two

model OVA-SLPs (OVA24 and OVA17) were introduced either co-encapsulated in the same particle

or loaded in separate liposomes, and their effect on the induction of antigen-specific T cells was

studied, also when combined with defined TLR ligands (Supplementary Table S2).

The obtained SLP-liposomes were positively charged, rather monodisperse and had an average size

below 200 nm (Supplementary Table S2). The bioactivity of the liposomal-loaded poly(I:C), Pam3CSK4

and CpG was analyzed in vitro by measuring the expression of the co-stimulatory marker CD86 on

the surface of DCs incubated with the formulations showing that loading of TLR ligands in the

current liposomes did not diminish their ability to activate DCs. Also, as shown previously (19), these

SLP-loaded cationic DOTAP-liposomes were able to promote DC uptake and processing of the SLP

antigen for MHC class I presentation, resulting in more efficient activation of SIINFEKL-specific CD8+ T

cells (B3Z cells), as compared to the free antigen (Supplementary Fig. S2).

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

The ability of OVA SLP-loaded cationic liposome formulations to induce cell-mediated immune

responses in vivo was tested by selecting poly(I:C) as the main TLR ligand adjuvant for a direct

comparison with our previous studies (19).

In mice immunized intradermally twice with the mixture of the separately loaded liposomes (OVA24-

lipos. +OVA17-lipos.) a high percentage of antigen-specific CD8+ T cells was detected, which was

elevated compared to the frequency in mice immunized with OVA24-liposomes only; indicating that




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OVA17-mediated help improved the CD8+ response. The response measured in the blood was

significantly higher in mice having been immunized with the adjuvant poly(I:C) formulation (OVA24-

lipos. + OVA17-lipos. + poly(I:C)) (Fig. 1, Supplementary Fig. S3). Liposomes co-encapsulated with

both SLPs showed increased T-cell numbers when poly(I:C) was included in the formulation, either

mixed with the SLP-liposomes (OVA24/OVA17-lipos. + poly(I:C)) or co-encapsulated with the

peptides (OVA24/OVA17/poly(I:C)-lipos.) (Fig.1).

In addition, all SLP-containing liposomal formulations induced high percentages (> 1.0 %) of

cytokine-producing CD8+ T cells and functional cytokine-producing CD4+ T cells were detected in all

groups treated with OVA17-containing liposomal formulations (Supplementary Fig. S4), indicating

that OVA17 retains its functionality when co-encapsulated in liposomes with OVA24.

The most potent formulations presented in Fig. 1 were evaluated for their capacity to induce

cytotoxic CD8+ T cells that could kill adoptively transferred target cells in vivo (Fig. 2). The activated

CD8+ T cells in mice immunized with SLP-containing liposomes exhibited strong target cell–specific

killing: vaccination with the mixture of separate SLP-loaded liposomes gave killing activity above

80%, and when poly(I:C) was included in the formulation, mixed or co-encapsulated with the SLPs,

the cytotoxic activity was even further enhanced (Fig. 2).

Similar results were obtained in mice vaccinated with the formulation where the poly(I:C) was

replaced by CpG in the mixture of the SLPs separately loaded in liposomes (Supplementary Fig. S5) or

Pam3CSK4, although the adjuvant effect in the latter case was less pronounced.

To conclude, our data indicate that the immunogenicity of SLP-loaded liposomes can be enhanced

with the incorporation of different TLR ligands. However, adjuvant formulations with poly(I:C)

tended to be more versatile , because the ligand in the presence of the TH SLP OVA17, could be

either co-encapsulated with the SLPs or simply mixed before immunization without compromising

the strength and the quality of the induced T-cell response.




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Liposome vaccine-mediated regression of established melanoma

The potential of our SLP-liposomal with poly(I:C) formulations as therapeutic antitumor vaccines

was investigated in an established melanoma model (Fig. 3). Nine days after the subcutaneous B16-

OVA tumor cell inoculation, when tumors had reached a significant palpable size, mice were injected

intradermally twice with the liposome vaccines in a two-week interval. Over a period of 60 days, all

mice in groups vaccinated with poly(I:C)-liposomal formulations could control the outgrowth of the

established tumors, whereas non-vaccinated mice or mice vaccinated with the free compounds (free

OVA24 + OVA17 + poly(I:C)) had to be sacrificed within three weeks because of progressively

growing tumors. In detail, mice vaccinated with the separate OVA24 and OVA17 liposomal mixtures

and poly(I:C) or the poly(I:C)-co-encapsulated SLPs (OVA24/OVA17/poly(I:C)-lipos.) showed a

substantial tumor regression with a delay of outgrowth of two to four weeks (Fig.3). No adverse

side-effects were observed at the injection spot. Additionally, when the OVA24/OVA17/poly(I:C)-

lipos. formulation was delivered intradermally at the tailbase instead of the abdominal area, a

significant three to five-week delay in outgrowth was observed. Two out of eight mice were even

cured of these aggressive tumors, indicating that vaccination effectiveness can be dependent on

location and drainage of the injection site.

Effective HPV E7-specific T-cell response induction by liposomes in vivo

Considering the promising results described above, the cationic liposomal formulations loaded with

the HPV E7-derived SLP were tested for their immunogenicity in vivo.

In blood of mice immunized with E7 SLP loaded into liposomes (E7-liposomes), a high percentage

(ca. 4%) of antigen-specific CD8+ T cells was detected a week after the second vaccination (day 21)

(Fig. 4A and Supplementary Fig. S6A). Similarly to the OVA SLP-loaded liposomes, the addition of

poly(I:C) in the formulation led to an even higher number: about 7% of the total CD8+ T cells were

E7-specific and the adjuvant effect of the poly(I:C) on the activation of CD8+ T cells was present,




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regardless the way the TLR3 ligand was loaded in the vaccine: co-encapsulation of the poly(I:C) with

the E7 SLP (E7/poly(I:C)-liposomes) or mixed with the E7-liposomes just before the immunization

(E7-liposomes + poly(I:C)) led to an equally strong induction of T cells. Furthermore, mice immunized

with the E7/poly(I:C)-liposomes intradermally on the tailbase, showed a trend to raise a higher

average number of activated T cells, compared to mice immunized at the abdominal area (Fig. 4A).

Ten days after the boost immunization (day 24), the number of the CD8+ T cells was analyzed also in

spleens of immunized mice, showing again significantly higher frequencies of antigen-specific T cells

for the groups having received adjuvant + liposomal vaccines, compared to the ones that received

the vaccine of the free compounds (free E7 + poly(I:C)) (Fig. 4B).

Analysis of blood samples and splenocytes with the H-2Db – RAHYNIVTF tetramer antibody revealed

the potency of the vaccines in the expansion of primed antigen-specific CD8+ T cells, while their

functionality was analyzed by cytokine production. Because the E7 SLP vaccine also contains a TH

epitope, we analyzed the induction of E7-specific CD4+ T cells after ex vivo re-stimulation of blood

samples from immunized mice collected on day 21 (one week after the boost immunization).

The intracellular cytokine analysis showed that all liposomal formulations induced high numbers of

IFNγ-producing CD8+ T cells, above 3 %, which correlates with the data of the RAHYNIVTF -tetramer

analysis. This percentage showed a two-fold increase when poly(I:C) was co-encapsulated with the

SLP (E7/poly(I:C)-liposomes), but not when mixed with the E7-liposomes (E7-liposomes + poly(I:C))

(Fig. 4C). Furthermore, E7-liposome vaccination induced activation of significant numbers of

functional E7-specific CD4+ T cells, producing IFNγ (Fig. 4E and Supplementary Fig. S6B). Activation of

cytokine-producing CD4+ T cells underlines the development of a TH1-oriented response,

characterized by the secretion of IFNγ and IL2 cytokines (Fig. 4F). With respect to that, a fraction of

IFNγ-producing CD8+ T cells also produced the effector cytokine TNFα (Fig. 4D), indicating that

specific CD4+ T cells were not only expanded by the vaccination, but also produced different

functional type 1 cytokines, further amplifying the effector T-cell immune response.




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As also shown for the OVA SLP-loaded liposomes, intradermal immunization of mice with 1 nmol of

E7 SLP-loaded into liposomes induced strong in vivo killing, above 70%, in contrast to vaccination

with free E7 (Fig. 5). Addition of poly(I:C) to the liposomal formulation (E7-liposomes + poly(I:C)),

however, did not further enhance cytotoxic activity. Vaccination with the E7 SLP/poly(I:C) co-

encapsulated liposomal formulation induced cytotoxicity up to 80% (Fig. 5).

In conclusion, the poly(I:C)–E7-liposome formulations efficiently induced a functional CD8 and CD4 T

cell-mediated immune responses. The E7 SLP-specific CD8+ T-cell frequency induced by the

liposomes showed a significant increase of the T-cell percentage as compared to the free E7 SLP

vaccines, with high in vivo killing capacity.

Adjuvant–E7 peptide-loaded liposomes efficiently eradicate established HPV16+ tumors

The therapeutic potency of the poly(I:C)–E7 SLP-liposome vaccines (containing 2.3 μg E7 SLP and 1

μg poly(I:C)) was investigated in mice bearing (s.c.) tumors induced by the TC-1 cell line, expressing

HPV16 E7 protein. As a control, the free compounds [free E7 SLP + poly(I:C)] were used as vaccines

in the same doses in PBS and emulsified in Montanide. The liposomal formulations were directly

compared to the clinical “gold standard” Montanide ISA-51 mixed with a 65-fold higher dose of E7

SLP and a 20-fold higher dose of poly(I:C) (high dose in Montanide: 150 μg and 20 μg, respectively)

(21).

Therapeutic treatment started nine days after TC-1 tumor cells inoculation when mice had clearly

palpable tumors. Non-treated mice, mice vaccinated with the free compounds vaccine [free E7 +

poly(I:C)], or mice injected with the low dose of vaccine in Montanide had to be sacrificed within

three weeks because of progressive tumor growth (Fig. 6). Within a period of 130 days, all mice in

groups vaccinated once with the poly(I:C)–E7 liposomal formulations or the standardized (high) dose

in E7-Montanide ISA-51 emulsion, could control outgrowth of the established TC-1 tumors. Mice

vaccinated intradermally with the E7/poly(I:C)-liposomes showed a substantial tumor regression




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with 5 out of 7 mice being completely cured from their established tumors. When the same

formulation was intradermally administered at the tailbase instead of the abdominal area, 100% of

the mice eradicated their tumors and were fully protected till the end of the experiment (Fig. 6).

Mice vaccinated with 2.3 μg of E7 SLP in non-adjuvanted liposomes (E7-liposomes) showed a delay

on the tumor outgrowth, comparable to that observed in mice injected with a 65-fold higher dose of

E7 SLP emulsified in Montanide ISA-51 and combined with a higher dose of poly(I:C) (Fig. 6 and

Supplementary Fig. S7A and B).

We observed a strong administration route–dependent effect on regression of tumor outgrowth,

with the intradermal vaccination being more efficient than the subcutaneous one (Fig. 6 and

Supplementary Fig. S7A and B). Additionally, although no significant difference was observed

between mice vaccinated intradermally at the abdominal area or the tailbase, when mice were

injected subcutaneously the different areas significantly influenced the tumor regression

(Supplementary Fig. S7A&B). Similarly, when the E7/poly(I:C)-liposomal formulation was delivered

subcutaneously, the tailbase injection seemed to be more potent than the abdominal one (50% and

12.5% cured mice, respectively), suggesting that vaccination effectiveness might depend not only on

the administration route, but also the injection site, as also observed in the B16-OVA model (Fig. 3).

In all mice vaccinated with liposomal formulations, no adverse side-effect was observed at the

injection spot, irrespective of the administration route or site; in contrast, the high-dose of

Montanide induced skin irritation on the shaved tailbase area of the immunized 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 of

their initial tumors were also fully protected by the new tumor inoculation. This observation

suggested that these mice raised functional memory T cells to control newly injected tumor cells




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(Fig. 6 and Supplementary Fig. S7C), as confirmed by high frequencies of functional antigen-specific

CD8+ T cells detected in blood at this time point (data not shown).

Finally, in order to be able to attribute the tumor regression to the induction of a E7 tumor–specific

T cell–mediated immunity, mice depleted from CD8+ T cells were vaccinated with the E7/poly(I:C)-

liposomal formulation via the most potent administration routes (i.d. abdominal area/ tailbase). All

mice that were CD8+ T cell-deficient had to be sacrificed within three weeks due to their tumors’ size

reaching the humane endpoint (Fig. 7A and Supplementary Fig. S7D), showing the crucial role of

CD8+ effector T cells in this system.

Non-depleted mice with very large tumors (> 400 mm3) treated intradermally with the E7/poly(I:C)-

liposomes, either at the abdominal area or at the tailbase, showed a remarkable tumor regression in

this experiment (Fig. 7B). In detail, 75% mice treated intradermally with one shot of E7/poly(I:C)-

liposomes were cured from their established tumors. Even when their tumors had reached a size of

about 500 mm3, a single vaccination could still induce full regression of large tumor masses, pointing

to the importance of the administration route effect and the potential of these cationic liposomes as

a therapeutic cancer vaccine system for SLPs.




16

Discussion

Here, the concept of cationic liposome-based formulations was investigated as the backbone for

improved delivery of SLPs harboring defined tumor antigens for active immunotherapy. SLPs have

gained much interest not only because of successful clinical trials with overlapping long peptides in

HPV-induced premaligant lesions, but also the importance of mutated antigenic sequences (neo-

epitopes) in several tumor types that can be readily produced as synthetic long peptides covering

the mutated area (22). This opens the opportunity for personalized specific immunotherapy, but it

requires optimal formulation for multiple and diverse SLP sequences.

We showed previously that a poly(I:C)–liposomal formulation induced a strong and long-lasting

cytotoxic T cell–mediated immune response against OVA24, an SLP antigen with relatively low

immunogenicity (19), and we reported the superior efficiency of liposomes when compared to other

SLP-loaded particulate systems (20). Even though the complexes of the MHC class I + peptide can be

recognized by naive CD8+ T cells directly, in order to become effector and cytotoxic T cells, they

require sufficient costimulatory signals from the activated DCs (23).

It is known that particulate delivery systems, including liposomes, offer numerous advantages for

DC-targeted vaccination in comparison with free antigens (24). The enhanced immunogenicity

observed for liposomal SLPs as compared to free SLPs is likely due to enhanced cellular uptake and

the capability of the DOTAP based liposomes to induce activation and maturation of DCs (19, 25). As

the developed formulation does not include any targeting molecule or device, the DCs are most

likely targeted in a passive way, for instance due to the small size and/or the cationic nature of the

liposomes. Moreover, as shown in the present study, cationic liposomes are excellent carriers for

defined TLR ligands, which retain their DC-maturing capacity after particle uptake. Regarding the

liposomes’ size, it has been suggested that small particles (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 by macrophages, leading to a humoral immune response (26,




17

27). However, some reported results are contradictory and the immunogenicity of liposomal

vaccines depends on multiple other factors (such as administration route) that need to be taken into

account (28).

Encouraging findings have been reported regarding the precise targeting of DCs upon intravenous

administration of non-functionalized RNA-lipoplexes (29). In the current study, using a rather low

dose of OVA or E7-derived long peptide (≤ 2.5 μg SLP/mouse) into cationic DOTAP-based liposomes,

we report high frequencies of SLP-specific CD8+ T cells as well as functional CD8+ and CD4+ T-cell

responses in mice, confirming the potency of positively charged liposome formulations. The way that

OVA-derived peptides, OVA24 and OVA17, were loaded into the liposomal formulation (co-

encapsulated or delivered in separate particles) did not compromise the induced immune response.

Finding suggested that multiple liposomes are most likely taken up by the same DC. Future confocal

microscopy studies using fluorescently-labelled liposomes and labelled SLP could give more insight

into the fate of the liposomes and the antigen following administration in vivo.

Activation and proliferation of CD4+ T cells is crucial for an optimal CD8+ T-cell response and

development of memory, (4) since CD4+ T cells help in the differentiation of naive CD8+ T cells into

effector T cells through activation signals, mostly IL2 secretion (30). Enhanced production of IFNγ

could also contribute to anti-HPV immunity (31), since IFNγ-associated T cell-responses are weak or

absent in patients with cervical cancer (32). So, based on the robust CD4+ T-cell responses detected,

the superiority of the liposomes over free SLPs as a cancer therapeutic vaccine can be shown.

Although the use of a TH SLP significantly improved CD8+ T-cell priming (33), the inclusion of a TLR

ligand adjuvant appeared to be crucial for the control of the tumor outgrowth. In both humans and

mice, a large list of TLR ligands is known and all of them function as adjuvants, stimulating DC

targeting and activation (25) and cross-presentation (23). In this study, the TLR3 ligand poly(I:C) was

the adjuvant most frequently combined 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,




18

was also achieved. Noteworthy, in contrast to several studies in which co-delivery of the adjuvant

with the antigen was beneficial (24, 34), we showed that the adjuvant effect of poly(I:C) was

independent of the way it was formulated: the co-encapsulation of poly(I:C) with the SLP in

liposomes or the mixture of the ligand with the SLP-liposomes are both strongly immunogenic

vaccines. Altogether, the results on the OVA-loaded liposomes’ characterization indicate not only

that is feasible to load two SLPs, either separately or co-encapsulated into liposomes, suggesting

that DCs can engulf multiple liposomes simultaneously, but also that the TLR-ligands can be replaced

and the peptide ratios can be adjusted to the desired final concentration, for better control and

easier adjustment of peptide ratios in multiple long-peptide vaccines.

The route of immunization is another crucial factor that can greatly influence the nature of the

induced T-cell response (35), considering the different cell types that can be targeted by just

changing the vaccine delivery route. Although many administration routes are under investigation,

the most commonly used route remains the subcutaneous (s.c.) one. Here we showed that a single

intradermal tailbase vaccination of TC-1 tumor-bearing mice with a low dose of E7 /poly(I:C)-

liposomes led to complete clearance of the tumors in 100% of the mice. Upon intradermal

vaccination, antigen presentation depends on the targeted APCs that transport the injected antigen

towards the draining lymph nodes (36) or the recruitment of DCs subsets in the local tissue or the

lymph nodes for promoting the cross-presentation of the antigen (37). Considering this, attractive

cellular target for vaccination approaches can be the CD8α+ DCs and their equivalents in tissues, due

to their enhanced ability to present exogenous antigen on MHC class I and their strong capacity to

provide a robust signal 3 (38). However, the contribution of skin-resident DCs to the liposomes’

immunogenicity cannot be excluded.

Cationic liposomes formulated with the appropriate TLR ligand and SLPs may be well-suited to

optimally target the most efficient APC via the intradermal route. Our results showing adequate

tumor eradication obtained with two independent SLP model systems using two aggressive tumor




19

types (TC-1 and B16 melanoma), suggest that cationic liposomes are not only an excellent delivery

platform for peptide-based therapeutic cancer vaccines, but also superior to the clinically used

Montanide ISA-51 emulsion or polymeric particulate delivery system, as also shown in our recent

comparative study (20). With respect to the latter, polymeric pLHMGA particles loaded with a

longer version of the HPV16 E7-derived SLP, showed a prolongation of mice survival (three weeks)

when used as therapeutic vaccine in the TC-1 model (39); however, no tumor eradication was

observed even when a high dose (i.e., 43-fold higher) of E7-loaded particles (100 SLP μg/mouse)

combined with poly(I:C) (50 μg/mouse, i.e., 50-fold higher than the dose in the current study) was

injected s.c. on the flank of tumor-bearing mice. Regarding the Montanide ISA-51 emulsion, the

exact adjuvant mechanism is not well understood; however, it is believed that it functions via a

sustained release from the local antigen depot. Considering the great efficiency of our liposomes, we

could conclude that internalization of particles may be more crucial for the induction of a cellular

immune response than the formation of a depot, in line with our recent observations with PLGA

particles (40).

Combination of liposomes for immunotherapy with other therapeutic strategies, such as

chemotherapy (particles loaded with doxorubicin, cisplatin (41, 42) or paclitaxel (43)) or

photodynamic therapy (PDT) can be expected to lead to better than the reported in literature result

(44).

In conclusion, adjuvant–cationic liposomes loaded with SLPs can be an efficient monotherapy

against established tumors in mice. SLP/poly(I:C)-loaded liposomes, when administered

intradermally, induce a strong T cell-mediated immunity, able to control the tumor outgrowth, as we

showed in two independent models (B16-OVA and TC-1). Poly(I:C)-adjuvanted E7-loaded-liposomes

cured 75-100% of the immunized mice from their large established tumors and they were proved to

be more effective than the clinically used Montanide formulation loaded with the standardized 65-

fold and 20-fold higher dose of E7 SLP and poly(I:C), respectively. The liposomes’ versatility regarding




20

the loading of different antigens and TLR ligands makes them a promising platform for SLP-based

immunotherapy of cancer.




21

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Figure legends

Fig. 1: Antigen (SIINFEKL)-specific CD8+ T cells responses in blood (day 21) and in splenocytes (day 24) following

intradermal immunization with 1 nmol of OVA24 SLP in different formulations on day 0 and 14. OVA specific

CD8+ T-cell responses were detected with SIINFEKL-H-2 Kb tetramers. The experiment was performed twice

with comparable results and were evaluated by one-way ANOVA with Bonferroni’s multiple comparison test,

* P < 0.05. Each dot represents the response of an individual mouse (n = 5, mean ±SEM).

Fig. 2: In vivo cytotoxicity assay. The mean percentages of the killing activity of OVA formulations are presented

based on the ratio of differentially labeled, transferred CD45.1+ specific target cells which could be detected in

splenocytes of mice immunized with 1 nmol of OVA SLPs-loaded formulations. Bar graph shows the mean

percentages of cell killing (+ SEM) on day 24. Data evaluated by one-way ANOVA with Bonferroni’s multiple

comparison test, *** P < 0.0001. Representative histograms of CFSE-labeled target cells: right peak=peptide

pulsed and left peak= control.

Fig. 3: Tumor outgrowth in a therapeutic melanoma model, in mice immunized i.d. twice (as the arrows

indicate) with different OVA SLPs formulations, after subcutaneous injection on day 0 with 1 x 105 B16-OVA

cells. Tumor sizes in individual mice (n = 8) (A) and survival plots (left: i.d. abdominal skin vaccinations, right:

tailbase i.d. vaccination) (B) are presented. The curve of the two OVA SLPs + poly(I:C) liposomal formulations

are significantly different from the free OVA SLPs + poly(I:C), *** P < 0.0001 calculated with the log-rank

(Mandel-Cox) test. The experiment was performed twice with comparable results.

Fig. 4: E7-specific CD8+ T cells responses in (A) blood (day 21) and (B) splenocytes (day 24) upon intradermal

immunization of five mice per group (at the abdominal area or tailbase) with 1 nmol of E7 SLP on day 0 and 14.

Frequencies of E7-specific CD8+ T cells were detected with RAHYNIVTF-MHC Db tetramer. The experiment was

performed twice with comparable results. Intracellular cytokine analysis in blood of immunized mice at day 21

(C-F). Blood samples were stimulated ex vivo overnight with the E7 SLP. Plots show CD8+ T cells producing IFNγ

(C); CD8+ T cells producing both IFNγ and TNFα (D); CD4+ T cells producing IFNγ (E); and CD4+ T cells producing

IL2 (F). Data shown are averages of 5 mice per group + SEM and were evaluated by one-way ANOVA with

Bonferroni’s multiple comparison test, *P < 0.05, ** P < 0.001 and *** P < 0.0001.

Fig. 5: In vivo cytotoxicity against RAHYNIVTF-presenting transferred target cells. The mean percentages of the

killing activity of E7 formulations are presented based on the frequency of the transferred CD45.1+ specific

target cells that could be detected in splenocytes of mice immunized with 1 nmol of E7 SLPs-loaded

formulations. Bar graph shows the mean percentages (+ SEM) on day 24. Representative histograms of CFSE-

labeled target cells: right peak = peptide pulsed and left peak = control. Data evaluated by one-way ANOVA

with Bonferroni’s multiple comparison test, *** P < 0.0001.




25

Fig. 6: Adjuvanted E7-liposomes efficiently eradicate established E7-specific tumors. Graphs depict tumor

outgrowth in a therapeutic model, in mice immunized i.d. or s.c. at day 9 after tumor inoculation (first arrow),

with different E7 SLP formulations, after they were subcutaneously injected on day 0 with 1x105 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 E7+ poly(I:C) high dose in Montanide, ** p< 0.005

evaluated with the log-rank (Mandel-Cox) test. The experiment was performed twice with comparable results.

Light grey arrow in both (A) and (B) plots indicated the tumor-rechallenge time point (D85).

Fig. 7: Tumor eradication is dependent on CD8+ T-cell immunity. Survival of WT mice immunized i.d. (arrow)

with the E7/poly(I:C) formulation, after having been subcutaneously injected on day 0 with 1x105 TC-1 cells, is

compared to WT mice with depleted CD8+ T cells (A). Eradication of established large tumors was observed in

WT mice immunized with the E7/poly(I:C) formulation intradermally on the abdominal area and tail base (B).




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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