A tumor-peptide based nanoparticle vaccine elicits efficient tumor … · 2019. 4. 6. · 1 A...

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A tumor-peptide based nanoparticle vaccine elicits efficient tumor

growth control in anti-tumor immunotherapy

Carolin Heße1, Sebastian Kollenda2, Olga Rotan2, Eva Pastille1, Alexandra

Adamczyk1, Christina Wenzek1, Wiebke Hansen1, Matthias Epple2, Jan Buer1, Astrid

M. Westendorf1, Torben Knuschke1*

1Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen,

Essen, Germany

2Institute of Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE),

Essen, Germany

Running title: Peptide nanoparticle vaccine for anti-tumor immunotherapy

Keywords: nanoparticles; therapeutic vaccine; tumor; T cell immunity; type I interferons;

checkpoint inhibition

*Corresponding author

Torben Knuschke, PhD

Institute of Medical Microbiology

University Hospital Essen

Hufelandstr. 55, 45122 Essen, Germany

Phone: +492017233093; Fax: +492017235602

e-mail: [email protected]

The authors declare no potential conflicts of interest.

Research. on December 7, 2020. © 2019 American Association for Cancermct.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 April 8, 2019; DOI: 10.1158/1535-7163.MCT-18-0764

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Abstract

Recognition of immunoactive oligonucleotides by the immune system, such as Toll-

like receptor ligand CpG leads to increased antibody and T cell responses. Systemic

application often results in unwanted generalized non-antigen specific activation of

the immune system. Nanoparticles are ideal carriers for small and large molecules.

Recently, we have demonstrated that calcium phosphate (CaP) nanoparticles

functionalized with CpG and viral antigens are able to induce specific T cell immunity

that protects mice against viral infection and efficiently reactivates the exhausted

CD8+ T cell compartment during chronic retroviral infection. Therefore, CaP

nanoparticles are promising vaccine vehicles for therapeutic applications. In this

study, we investigated the therapeutic potential use of these nanoparticles in a

murine xenograft colorectal cancer model. Therapeutic vaccination with CaP

nanoparticles functionalized with CpG and tumor model antigens increased the

frequencies of cytotoxic CD8+ T cells in the tumor in a type I interferon (IFN I)

dependent manner. This was accompanied with significantly repressed tumor growth

in contrast to the systemic administration of soluble CpG and antigens. Combination

therapy of CaP nanoparticles and immune checkpoint blocker against PD-L1 further

enhanced the cytotoxic CD8+ T cell response and eradicated the tumors. Strikingly,

vaccination with CaP nanoparticles functionalized with CpG and the primary tumor

cell lysate was also sufficient to control the tumor growth. In conclusion, our results

represent a translational approach for the use of CaP nanoparticles as a potent

cancer vaccine vehicle.

Introduction

Induction and strengthening of CD8+ and CD4+ T cell responses is a major goal of

immunotherapy. Particularly cytotoxic CD8+ T cell responses are important to exert

protective effects against uncontrolled tumor growth. These T cells are primed by

recognizing small, antigenic peptides that are processed and presented by antigen

presenting cells (APCs) on MHC molecules (1). Especially dendritic cells (DCs) as

part of the first line of immune defense play an important role in this regard. Distinct

peptide epitopes then activate specific T cell clones. Consideration of highly

immunogenic, cancer specific epitopes for therapeutic vaccination, so called

neoantigens, could therefore enhance the efficacy of tumor cell eradication but need

to be tailored for each individual patient (2). However, peptide-based vaccination




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approaches typically generate weak cellular responses due to a short half-life of the

peptides in vivo and insufficient stimulation of innate immune components in an

immune resistant tumor environment (3,4). Suitable transportation of biomolecules

and additional stimuli are necessary for efficient innate and adaptive immune

responses against the tumor. Recent studies and clinical trials confirmed the potential

of different immunotherapeutic agents that can break immune tolerance in cancer

patients (5-9). However, the clinical response was rather poor since only a small part

of patients were free of disease after treatment (10). Thus, vaccine vehicles are

needed that stimulate adaptive immunity appropriately. We have recently described a

calcium phosphate (CaP) based nanoparticle system for vaccine purposes (11).

Multishell CaP nanoparticles are ideal carriers for biomolecules as they can transport

many molecules across the cell membrane and protect the encapsulated

biomolecules against enzymatic degradation (12-14). After endocytosis of

functionalized CaP nanoparticles they are dissolved in lysosomes and the

encapsulated biomolecules are released and processed by DCs (15,16). In

combination with CpG and viral antigens CaP nanoparticles facilitate a strong

activation of DCs and generation of virus-specific T cells (17). Furthermore,

application of functionalized CaP nanoparticles during chronic viral infection was

sufficient to overcome barriers of T cell exhaustion and supported the reinforcement

of cytotoxic CD8+ T cells in contrast to the administration of soluble CpG and

peptides (18,19). However, combination immunotherapy might further improve the

outcome of the disease. In particular, combination of agents targeting inhibitory

pathways such as the programmed cell death protein-1 (PD-1) is under investigation

to enhance the effects of immunotherapy (20). By blocking PD-1 or its ligand PD-L1,

exhausted cytotoxic T cells can regain their function and eradicate effectively tumor

cells.

Here, we explored the efficacy of CpG and tumor associated antigen (TAA)

functionalized CaP nanoparticles for therapeutic cancer treatment by inducing strong

tumor-specific CD8+ T cell immunity. By taking advantage of a murine xenograft

tumor model expressing the viral antigen hemagglutinin (HA), we demonstrated that

the administration of HA-peptide and CpG functionalized CaP nanoparticles was

highly sufficient to enhance the anti-tumor T cell response and to repress tumor

progression. This effect was strongly dependent on the induction of type I interferons

(IFN I), which boosted the cytotoxicity of CD8+ T cells. Importantly, we demonstrated




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that combination therapy of functionalized CaP nanoparticles and PD-1 blockade

increased this anti-tumor effect. Additionally, instead of single model antigens we

loaded the particles with a whole peptide pool derived from a primary tumor cell

lysate representing a generalized approach for individual cancer therapy. Since also

here the treatment with functionalized CaP nanoparticles of tumor-bearing mice led to

significantly decreased tumor growth, we propose a high therapeutic potential of

functionalized CaP nanoparticles for the immunotherapeutic treatment of cancer.

Material and Methods

Mice

BALB/c mice were purchased from Envigo Laboratories (Envigo CRS GmbH). CL4-

TCR transgenic mice express an α/β-TCR specific for an MHC-I-restricted epitope of

HA (H-2Kd:HA512–520) on CD8+ T cells (21). All mice used in the experiments were 8

to 10 weeks old and housed under specific pathogen-free conditions in the

Laboratory Animal Facility of the University Hospital Essen. This study was carried

out in accordance with the recommendations of the Society for Laboratory Animal

Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory

Animal Science Associations (FELASA). The protocol was approved by the North

Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection

(LANUF), Germany (Permit Number: Az.: 84-02.04.2014.A290).

Cells and cell culture

The CT26 colon cancer cell line was obtained from the American Type Culture

Collection (Manassas). CT26 cells were maintained in Iscove’s Modified Dulbecco’s

Medium (IMDM) containing 10% endotoxin free fetal calf serum (FCS), 50 µg mL-1

penicillin/streptomycin and 25 µM β-Mercaptoethanol. Cell lines were maintained in a

humidified 5% CO2 atmosphere at 37°C. Cell vials were stored in liquid nitrogen and

passaged twice before injection. Mycoplasma testing was performed every 2 months

by PCR on in vitro propagated cultures. No additional authentication method was

performed.

To generate HA expressing CT26 cells the cDNA sequence of HA was cloned into

the pEF/myc vector containing a neomycin resistance cassette and in which HA is

constitutively expressed under the control of an EF1α-Promotor. CT26 cells were




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then transfected with the pEF/myc-HA vector using the TurboFectTM cell transfection

reagent (Thermo Fischer Scientific), according to the manufacturer’s instruction.

Transfected cell were selected by adding 600 µg/ml Geneticin (G418) (Gibco) to the

culture medium and cultured for 14 days.

TLR-ligand and peptides

The phosphorothioate-modified class B CpG 1826 was purchased from Eurofins

MWG Operon. Influenza derived peptides (strain A/PR/8/34) had the following

sequences containing MHC class II and MHC class I epitopes: HA110–120,

SVSSFERFERFEIFPKESS;

HA512–520, YQILAIYSTVASSLVLL (Intavis AG).

Preparation of functionalized nanoparticles

The preparation of triple-shell nanoparticles functionalized with CpG and the HA110-120

or HA512-520 peptides was described previously (11,17,22). According to this synthesis

nanoparticles loaded with tumor proteins of a whole-cell lysate were prepared. Here

the tumor proteins served as cargo instead of the peptides. Briefly, 50 µL of the

HA110-120 or HA512-520 peptide (1 mg mL-1) or tumor proteins (1 mg mL-1), followed by

0.5 mL of calcium nitrate solution (6.25 mM) and then 0.5 mL of diammonium

hydrogen phosphate solution (3.74 mM) were added to the dispersion of single-shell

nanoparticles, i.e. CaP/CpG, leading to the triple-shell nanoparticles CaP/CpG/HA512-

520/CaP/CpG, CaP/CpG/HA110-120/CaP/CpG, CaP/CpG/tumor proteins/CaP/CpG),

respectively.

Next, the triple-shell nanoparticles were removed from the supernatant by

ultracentrifugation for 30 min at 66,000 g. The concentration of the incorporated

biomolecules was determined by UV/Vis spectroscopy on the supernatant in a DS-11

FX+ Nanodrop instrument (DeNovix). The final concentrations of CpG, HA110-

120/HA512-520 peptide and tumor proteins were 20.6 µM (125 µg mL-1), 41 µg mL-1 and

50 µg mL-1, respectively. Then, the centrifuged NPs were redispersed in water

(typically in 1 mL) with an ultrasonic processor (UP50H, Hielscher, Ultrasound

Technology; cycle 0.8, amplitude 60%) for 10 s.

All inorganic salts were of p.a. quality. Ultrapure water (Purelab ultra instrument from

ELGA) was used for all preparations. All formulations were prepared and analyzed at

room temperature and sterile filtrated before usage. The particles were characterized




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by scanning electron microscopy (ESEM Quanta 400) with palladium-gold sputtered

samples. Dynamic light scattering was performed with a Zetasizer nanoseries

instrument (Malvern Nano-ZS, λ = 532 nm). The particle size data refer to scattering

intensity distributions (z-average). Calcium concentrations were determined by

atomic absorption spectroscopy (AAS; M-Series AA spectrometer; ThermoElectron

Corporation, Schwerte, Germany) after dissolution of the particles in hydrochloric

acid.

Scanning electron microscopy showed spherical NPs with a typical diameter of 75 to

80 nm. For peptide-carrying nanoparticles, dynamic light scattering gave a

hydrodynamic radius of 100 nm, indicating a good dispersion. The negative zeta

potential of −18 mV was due to the outer shell of anionic CpG. For the tumor protein-

carrying nanoparticles, dynamic light scattering gave a hydrodynamic radius of 190

nm (indicating a low degree of agglomeration) and a zeta potential of −18 mV. The

calcium concentration in the dispersions was 23.2 μg mL−1 Ca2+ for the peptide-

carrying particles and 22.3 μg mL−1 Ca2+ for the tumor protein-carrying particles (by

AAS), resulting in estimated calcium phosphate concentrations of 58 and 56 μg mL−1,

respectively (assumption of the stoichiometry of hydroxyapatite; Ca5(PO4)3OH).

Together with the particle diameter from SEM (75 to 80 nm), this gives a particle

concentration of 7.3x109 particles per mL-1. With these data, the mass ratios of

biomolecule (CpG, HA peptides and tumor proteins) per CaP nanoparticle were

calculated. For the peptide-carrying particles, the mass ratios were 2.15:1 =

CpG:CaP and 0.70:1 = peptide:CaP. In case of the protein-carrying particles, the

ratios were 2.23:1 = CpG:CaP and 0.89:1 = protein:CaP.

Tumor cell lysate preparation

CT26 cell pellets were washed and suspended in phosphate buffered saline (PBS)

and subjected to four freeze-thaw cycles. Finally, cell death and lysis were confirmed

by Trypan blue exclusion and cell were centrifuged at 16,300g for 5 minutes to

remove cellular debris. Supernatants were collected and stored at -20°C. Protein

content of lysate preparation was measured using a Bicinchonic acid (BCA) protein

assay kit (Pierce) according to manufacturer’s protocol.

Antibodies and flow cytometry




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The monoclonal antibodies αCD4 (clone RM4-5), αCD8 (clone 53-6.7), αCTLA-4

(clone UC10-4F10-11) and αCD43 (clone 1B11) were obtained from BD Biosciences.

αGzmB antibody (clone GB12, Invitrogen) was used for intracellular granzyme B

staining. The αFoxp3 (clone FJK-16s), αKlrg1 (clone 2F1), αCXCR3 (clone CXCR3-

173) and αKi67 (clone SolA15) antibody was purchased from eBioscience. αTIM-3

(clone 215008) antibody was purchased from R&D. Intracellular staining with αKi67,

αFoxp3, αCTLA-4 and αGzmB was performed as described previously (18). Data

was acquired by using an LSR II instrument using DIVA software (BD Biosciences).

Tumor repression by adoptive transfer of HA-specific T cells

5x105 CT26 or CT26-HA tumor cells were s.c. transplanted into the right or left flank

respectively. Additionally 106 HA-specific CD8+ T cells were adoptively transferred via

i.v. injection at day 3, 7 and 10 post tumor cell injection. Tumor volumes were

calculated until day 12 post tumor transplantation.

In vivo tumor transplantation

Mice were injected subcutaneously (s.c.) with 105 viable CT26 or CT26-HA tumor

cells in Matrigel (Corning). Tumor volumes were calculated using following formula:

(length × width × height).

Therapeutic vaccination of mice

Tumor bearing mice were vaccinated at day 3, 5 and 7 post tumor transplantation

s.c. either with PBS or CaP NPs functionalized with CpG/HA (10.3 µM CpG and 41

µg mL-1 HA110-120/HA512-520 peptide; CaP/CpG/HA/) or soluble CpG and HA peptides

(CpG/HA) in both hind footpads (50 µL each, i.e. 3.6x108 particles per dose). 12 days

post tumor transplantation tumors and tumor draining lymph nodes were harvested.

Cells of lymph nodes and tumors were isolated and used for further analysis.

Isolation of cells from lymph nodes and tumors

Lymph node (LN) cells were meshed through a 70-μm cell strainer and washed with

PBS containing 2 mmol/L EDTA and 2% FCS. Tumors were meshed through a 70-

µm cell strainer and washed with IMDM containing 10% FCS, 50 µg mL-1

penicillin/streptomycin and 25 µM β-mercaptoethanol.




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Depletion of CD8+ T cells

For the depletion of CD8+ T cells during the vaccination of mice, 200µg anti-mouse

CD8 depleting antibody (clone 53-6.7, BioXcell) was injected intraperitoneally on day

3, 5 and 7 after tumor cell transplantation.

IFNAR blockade

To block IFN I signalling, mice were injected intraperitoneally with 250µg anti-mouse

IFNAR-1 blocking antibody (clone MAR1-5A3, BioXcell) diluted in PBS one day

before and 2 days after vaccination with functionalized CaP nanoparticles.

PD-1 blockade

To block PD-1 signalling, mice were injected intraperitoneally with 200µg anti-mouse

PD-L1 blocking antibody (clone 10F.9G2, BioXcell) diluted in PBS with or without

combination of CaP nanoparticle vaccination on day three and six after tumor cell

transplantation.

Statistical Analysis

Statistical analysis was performed by using student’s t-test or One-way ANOVA to

compare multiple groups using Tukey’s multiple comparison test. Data analysis was

performed using Prism 6.0 software (GraphPad). Statistical significance was set at

the level of p<0.05.

Results

Therapeutic treatment with CpG and tumor antigen functionalized CaP

nanoparticles represses tumor growth

To test the efficacy of functionalized CaP nanoparticles for the induction of potent

CD8+ T cell responses in cancer immunotherapy, we took advantage of a xenograft

tumor model. To demonstrate the CD8+ T cell-dependent tumor growth repression we

transplanted CT26 colon tumor cells, expressing the influenza virus derived protein

HA (CT26-HA) as a model antigen, subcutaneously (s.c.) into the right flank of mice.

Tumor growth was highly repressed by the adoptive transfer of HA-specific CD8+ T

cells, isolated from T cell receptor (TCR) transgenic CL4-TCR mice. In contrast CT26

tumors injected into the left flank were not affected (Fig. 1A). Thus, a profound tumor-




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specific CD8+ T cell response has been induced resulting in tumor growth control.

Next, we treated CT26-HA tumor bearing mice s.c. with functionalized CaP

nanoparticles containing CpG and MHC I and MHC II restricted epitopes of HA on

days 3, 5 and 7 after tumor cell transplantation (Fig. 1B). To see the advantage of the

nanoparticle system over systemic application of CpG in combination with the HA

peptides, mice were also treated with the same agents in dissolved form. In line with

the transfer of HA-specific CD8+ T cells, tumor growth was significantly delayed in

CaP nanoparticle treated mice compared to untreated mice (Fig. 1C). Treatment with

functionalized CaP nanoparticles was also significantly more efficient in inhibiting

tumor growth than the soluble administration of CpG and antigens. These results

indicate that the therapeutic vaccination with CpG and tumor-peptide functionalized

CaP nanoparticles might substantially boost adaptive immunity.

Therapeutic treatment with CpG and tumor antigen functionalized CaP

nanoparticles increases T cell activation

To identify the immunological mechanisms of tumor growth control after CaP

nanoparticle vaccination, we isolated and analyzed cells from the tumor draining

lymph node on day 12 after tumor cell transplantation. Interestingly, the therapeutic

vaccination with functionalized CaP nanoparticles did not increase the frequencies of

CD8+ or CD4+ T cells nor Foxp3+ regulatory T cells (Treg) compared to PBS or CpG

and HA treated mice (Fig. 2A). However, CD8+ T cells were strongly activated in both

treated groups (Fig. 2B). CD8+ T cells expressed significantly enhanced levels of the

proliferation marker Ki67 after nanoparticle vaccination. More importantly,

frequencies of granzyme B (GzmB) expressing, fully activated CD43+ CD8+ cytotoxic

T cells were also significantly increased. In line with this, the characteristic

transcription factor of cytotoxic CD8+ T cells eomesodermin (EOMES) (23) was

significantly elevated only in CD8+ T cells of mice that received CpG and tumor-

peptide functionalized CaP nanoparticles. These findings suggest that the

therapeutic vaccination with functionalized CaP nanoparticles repressed tumor

growth by enhanced activation of cytotoxic CD8+ T cells.

To understand the underlying anti-tumoral effect in more detail, CD8+ T cells

infiltrating the tumor were analyzed. In contrast to the consistent frequencies of

cytotoxic CD8+ T cells in the lymph nodes in all groups, vaccination with

functionalized CaP nanoparticles significantly enhanced the frequencies of CD8+ T




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cells infiltrating the tumor. At the same time no increase in CD8+ T cell frequencies

was detected after CpG/HA treatment (Fig. 3A). Importantly, frequencies of CD4+ T

cells infiltrating the tumor were strongly decreased compared to unvaccinated mice

suggesting that CD8+ T cells seem to be the main players for controlling the tumor

growth. Of note, no changes in the frequencies of Foxp3+ regulatory CD4+ T cells

were observed in any of the immunized groups (Fig. 3A). Consistent with the results

obtained from the draining lymph node, CD8+ T cells isolated from tumors were

strongly activated, indicated by enhanced Ki67 expression, expression of GzmB (Fig.

3B) or the terminal differentiation marker Klrg1 (Fig. 3C) although there was no

significant difference between the soluble CpG and functionalized nanoparticle

group. However, the expression of CD69 was significantly elevated after CaP

nanoparticle vaccination compared to soluble CpG/peptide administration. On the

other hand, while no changes in the expression of the inhibitory molecule PD-1 was

observed, TIM-3 expression was significantly upregulated after administration of

soluble CpG and antigens (Fig. 3D). To identify why CD8+ T cells migrate more

efficiently into tumors after CaP nanoparticle vaccination, we analyzed the expression

of chemokine receptors on CD8+ T cells. CXCR3, CCR4 as well as CCR5 were only

significantly increased on CD8+ T cells from nanoparticle treated mice (Fig. 3E).

Interestingly, the frequency of cytotoxic CD8+ T cells was strongly connected with the

tumor growth, since high frequencies of GzmB expressing CD8+ T cells correlated

with smaller tumor volumes in CaP nanoparticle-treated mice at day 12 after tumor

transplantation (Fig. 3F). Indeed, depletion of CD8+ T cells during vaccination of mice

abolished the anti-tumor effect of the CaP nanoparticle vaccination (Fig. 3G).

Therefore, these results suggest that CaP nanoparticle vaccination promotes the

infiltration of the tumor by cytotoxic CD8+ T cells, which are predominantly

responsible for the repression of tumor growth.

Type I IFNs are essential for the anti-tumor response induced by CaP

nanoparticle vaccination

Recently, we have demonstrated that reactivation of virus-specific CD8+ T cells with

functionalized CaP nanoparticles is dependent on IFN I during therapeutic

vaccination of chronically infected mice (24). In this study, CpG was identified as

driving force of IFN I release. To dissect the mechanism of how cytotoxic CD8+ T

cells are induced during therapeutic vaccination of tumor bearing mice, we treated




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the mice with an interferon alpha receptor I (IFNAR) blocking monoclonal antibody in

addition to CaP nanoparticle immunization (Fig. 4A). As expected, therapeutic

vaccination with CaP nanoparticles led to a significantly repressed tumor growth

compared to mice treated with PBS. In contrast, in mice treated with anti-IFNAR

antibody in addition to CaP nanoparticles the anti-tumoral effect was abolished. No

difference in tumor volume was observed when mice were treated with PBS and anti-

IFNAR antibody (Fig. 4B). Consistent to our previous results, vaccination with CaP

nanoparticles led to significant stronger expression of Ki67 and GzmB by CD43+

CD8+ T cells in tumor draining lymph node and tumors after CaP nanoparticle

immunization compared to unvaccinated mice (Fig. 4C&D). However, this effect was

abolished when IFNAR was additionally blocked during vaccination. Interestingly,

unresponsiveness to IFN I diminished frequencies of CD8+ T cells in the tumor and

led to significantly lower frequencies of cytotoxic CD8+ T cells at the tumor site

compared to CaP nanoparticle vaccination. These findings underline the importance

of IFN I for the efficient activation of cytotoxic CD8+ T cell dependent eradication of

tumor cells during therapeutic vaccination.

Combination therapy of CpG/tumor antigen functionalized CaP nanoparticle

vaccination and PD-1 blockade enhances the CD8+ T cell mediated anti-tumor

response

Blockade of the inhibitory PD-1/PD-L1 pathway reinforces CD8+ T cell responses

during infectious disease or cancer. To test whether we could further enhance the

anti-tumor effect mediated by vaccination with functionalized CaP nanoparticles, we

combined therapeutic vaccination with the blockade of PD-L1 (Fig. 5A). Injection of a

PD-L1 blocking monoclonal antibody inhibited the tumor growth comparable to the

therapeutic vaccination with functionalized CaP nanoparticles (Fig. 5B). Importantly,

the strongest reduction in the tumor volume was detected by the blockade of the PD-

1 pathway and vaccination with CaP nanoparticles. Approximately 30% of the mice

receiving CaP nanoparticles and a PD-L1 blocking antibody were tumor free on day

12 post tumor cell transplantation. Combination therapy significantly enhanced the

proliferation of CD8+ T cells compared to anti-PD-L1 or CaP nanoparticle treatment

alone in the lymph node, indicated by Ki67 expression (Fig. 5C). In line with this,

CD8+ T cells showed higher expression of Ki67 in the tumor after combination

therapy compared to nanoparticle treatment alone. Especially the frequency of GzmB




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expressing cytotoxic CD8+ T cells in the tumor was significantly elevated compared to

CaP nanoparticle vaccination or anti-PD-L1 treatment alone (Fig. 5D). Therefore, the

combination of therapeutic CaP nanoparticle vaccination with blockade of PD-L1

additionally strongly enhances the CD8+ anti-tumor response to the point of tumor

eradication.

Encapsulation of tumor lysate decreases tumor growth

Our results demonstrate the effectiveness of CpG and tumor-peptide functionalized

CaP nanoparticles for the therapeutic treatment of cancer. However, the used

antigens were based on the model antigen HA, expressed by the CT26 tumor cells.

For clinical application the identification of immunogenic tumor associated

neoantigens specifically tailored for the patient would be necessary. Because of the

high degree of polymorphism of human leukocyte antigens and issues of tumor

escape from the immune response, an easier approach to overcome the barrier of

unknown TAA would be to load nanoparticles with whole tumor cell lysates,

containing the entire peptide repertoire of the respective tumor of the patient (25-27).

Therefore, we tested the clinical applicability of CaP nanoparticles by functionalizing

them with a lysate of primary CT26 tumor cells in combination with CpG. Using this

approach allows to sensitize a wide range of tumor-specific T cells. Mice were

transplanted with CT26 tumor cells s.c. and therapeutically vaccinated with CaP

nanoparticles containing CpG and HA peptides or a whole tumor peptide pool from a

cell lysate. Therapeutic vaccination of tumor bearing mice with CpG and tumor lysate

delivered via CaP nanoparticles repressed the tumor growth significantly (p<0.0001,

Fig. 6A). Also the vaccination with soluble CpG and lysate had a significant effect

(p<0.01) although it induced only a 1.9 fold decrease of the tumor volume compared

to 3.2 fold decrease after CaP nanoparticle vaccination. Importantly, neither

immunization of CT26 tumor bearing mice with CpG and HA containing nanoparticles

nor soluble CpG and HA significantly altered the tumor growth which underlines the

necessity of tumor associated antigens within the vaccine. As before, CD8+ T cells in

the tumor draining lymph nodes and tumors were analyzed on day 12 post tumor cell

transplantation. Therapeutic vaccination with functionalized CaP nanoparticles

significantly enhanced the proliferation of CD8+ T cells in the tumor draining lymph

node of mice compared to PBS or soluble CpG/tumor lysate treatment (Fig. 6B). We

also noted a significant increase in GzmB expressing fully activated CD43+ cytotoxic




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CD8+ T cells in the lymph nodes and tumors of CaP nanoparticle immunized mice

accompanied by elevated frequencies of CD8+ T cells at tumor sites (Fig. 6C). These

results demonstrate that the induced anti-tumoral effects are strongly dependent on

the antigens used in the vaccine. Moreover, functionalization of CaP nanoparticles

with a tumor lysate is highly effective in inducing tumor specific cytotoxic CD8+ T cell

immunity, which dampens tumor growth, and therefore impressively underlines the

flexibility of the CaP nanoparticle system in this translational approach.

Discussion

Peptide based vaccines gain more and more importance for the therapeutic

treatment of cancer. Using tumor-specific T cell epitopes activates and expands T

cells that are capable of strong tumor eradication. However, recent clinical trials

revealed that induced immune effects using conventional approaches elicit various

effects so that only a low frequency of patients was free of disease (10,28-30). In

contrast, new vaccine formulations might help to enhance the efficiency of tumor

therapy (31,32). Some of the challenges that therapeutic tumor vaccines have to

overcome are for example the low immunogenic microenvironment, poor DC

activation and tumor heterogeneity (33). However, inclusion of tumor associated

antigens into novel vaccine formulations could enhance the success of tumor therapy

(34). Therefore, we have established use of CpG and tumor antigen functionalized

CaP nanoparticles for the therapeutic treatment of cancer. Both, using model

antigens expressed by a transfected tumor cell line as well as tumor derived peptides

from tumor lysates, significantly repressed tumor growth in a xenograft mouse model.

Importantly, this was not the case using CpG and tumor peptides alone. The current

findings illustrate that this effect was strongly dependent on the activation of cytotoxic

CD8+ T cells. Moreover, therapeutic vaccination with functionalized CaP

nanoparticles was much more efficient in activating these cells in the tumor draining

lymph nodes and tumors as compared to the systemic application of CpG and

antigens. One reason for this positive effect could be that the co-encapsulation of

CpG and tumor antigen may accelerate the uptake by DCs and reinforce innate and

adaptive immunity as previously suggested by others (35,36). In this line, we have

recently demonstrated that DCs are predominantly targeted in vivo by CpG and viral

antigen functionalized CaP nanoparticles and induce protective virus-specific CD8+ T

cell immunity (18). Further, the encapsulation may also extend the maintenance of




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CpG and tumor antigens in vivo. In the current study, we demonstrate that

immunization with functionalized CaP nanoparticles also enhances CD8+ T cell

activation and infiltration into tumors, which correlates with a repressed tumor growth.

Furthermore, a guaranteed co-delivery of CpG and antigens was shown to increase

the survival of antigen-specific CD8+ T cells since the vaccination with tumor antigens

alone enhance tumor growth, associated with Fas and PD-1 dependent apoptosis

induction in CD8+ T cells (37). CpG itself is already an established adjuvant (38). It

directly activates TLR9 expressing DCs, macrophages and B cells and therefore

contributes to the activation of both, innate and adaptive immune responses by

inducing Th1 cytokines (including IFN-gamma and IL-12) which improves the CD8+ T

cell response (39). As a consequence, we found that tumor infiltrating CD8+ T cells

are more activated after CaP nanoparticle vaccination compared to soluble

administration of CpG and antigens. The more efficient infiltration of the tumor by

CD8+ T cells after nanoparticle vaccination could be explained by the higher

induction of chemokine receptor expressions of CCR4 and CCR5, which have been

correlated with enhanced T cell infiltration and survival before (40). Although CCR4 is

also known to attract Tregs into the tumor microenvironment, it could also direct the

interaction between CCR5+ CD8+ T cells and DCs within the tumor (41). We recently

demonstrated that the TLR9 mediated release of IFN I leads to a profound activation

of the cytotoxic CD8+ T cell compartment during chronic viral infection (24). Here, we

describe that IFN I, induced by therapeutic vaccination, is crucial for the successful

eradication of tumors. Without the ability of the cells to respond to IFN I by the

application of an IFNAR blocking antibody, frequencies of cytotoxic CD8+ T cells

dramatically decreased in tumors and thus, abrogated the repressed tumor growth.

These results correlate with previous studies that point out the importance of IFN I for

the efficiency of tumor vaccines (32,36). Although we did not investigate the anti-

tumoral effects of immunization induced IFN I on other cell subsets, such as

neutrophils (42) or NK cells (43), we were able to demonstrate that an induction of

tumor-specific CD8+ T cells is essential for the efficient control of tumor growth since

the depletion of CD8+ T cells or immunization of mice bearing non-HA expressing

CT26 tumors with HA functionalized CaP nanoparticles only had minor effects on

tumor growth. Importantly, unspecific immunization did not lead to a decrease in

tumor volume suggesting that a direct effect of IFN I on the tumor cells can be

excluded since it was demonstrated that IFN I can upregulate p53 in tumor cells (44).




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Tumor-specific effector T cells can be highly suppressed and affected in their

functionality by the tumor microenvironment (45). Checkpoint inhibitors such as

CTLA-4, PD-1 or PD-L1 targeting monoclonal antibodies can restore T cell functions

and are approved for clinical cancer treatment (46). However, patients respond

differently to immune checkpoint therapies. Thus, therapies combining immune cell

targeting antibodies and vaccines pushing the anti-tumor immune response are

under extensive investigation (20). Our results demonstrate that the therapeutic

vaccination with CpG and tumor antigen functionalized CaP nanoparticles is very

efficient in inhibiting tumor growth compared to the soluble application of CpG and

tumor antigens. More importantly, we show that this effect can be increased by a

combination therapy consisting of CaP nanoparticle vaccination and blockade of the

inhibitory PD-1 pathway to fully control tumor growth. Blockade of PD-L1 enhanced

the effector function of cytotoxic CD8+ T cells indicated by enhanced GzmB

expression. Interestingly, the combination with therapeutic vaccination with CaP

nanoparticles had an additive effect as more GzmB+ CD43+ CD8+ effector T cells

were detectable in tumors. Since CT26 is a very aggressive colon carcinoma cell line

(47) our results therefore reflect the importance of reinforcement of cytotoxic CD8+ T

cells by immune therapy to control tumor growth.

Beside the application of distinct HLA restricted tumor derived T cell epitopes another

approach using whole tumor cell lysates generalizes the therapeutic treatment of

cancer since it covers an entire range of prospective tumor targets. In our

experimental setting we tested the functionalization of CaP nanoparticles with

primary tumor cell lysates. Using this approach makes it unnecessary to identify

specific immunogenic tumor associated antigens recognized by T cells, which is

rather difficult because of the high degree of polymorphism of human leukocyte

antigens (48). However, one important issue of tumor lysate based vaccines is to

overcome the tolerance against potential tumor associated self-antigens. The

immunization with CpG and tumor cell lysate functionalized CaP nanoparticles

significantly enhanced the proliferation of CD8+ T cells in the tumor draining lymph

node and enhanced frequencies of cytotoxic CD8+ T cells compared to PBS or

soluble CpG/tumor cell lysate treatment. Unlike most prior studies, in which DCs

were loaded with soluble lysate preparations and failed to induce potent immune

activation, immunization with CpG and tumor cell lysate functionalized CaP




16

nanoparticles efficiently facilitated to overcome immune tolerance which is often seen

in patients with solid organ malignancies (49-51).

In conclusion, we here demonstrate that CpG and antigen functionalized CaP

nanoparticles have a high potential for therapeutic vaccination against tumor disease.

Delivery of these molecules by CaP nanoparticles is more efficient than conventional

application of soluble CpG/antigen to repress tumor growth. This effect strongly

correlates with the reinforcement of cytotoxic CD8+ T cell immunity. A combination

therapy including PD-L1 blockade leads to a high effective tumor growth control.

Furthermore, we present a translational approach for the application of CaP

nanoparticles as a potent cancer vaccine vehicle by encapsulating a primary tumor

cell derived lysate.

Acknowledgements

T. Knuschke, A.M. Westendorf, J. Buer and W. Hansen were supported by the

Deutsche Forschungsgemeinschaft (DFG)-funded research training group RTG

1949.

We thank Svenja Groten, Christina Liebig, Christian Fehring and Mechthild Hemmler-

Rohloff for excellent technical assistance.

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Figure Legends

Figure 1: Therapeutic vaccination with CpG and tumor-peptide functionalized CaP

nanoparticles represses tumor growth. A) 5x105 CT26 or CT26-HA tumor cells were

s.c. transplanted into the right or left flank respectively. Additionally 106 HA-specific

CD8+ T cells were adoptively transferred at day 3, 7 and 10 post tumor cell

transplantation. Tumor volume was measured at indicated time points. n = 5 – 8.

Statistical analysis was performed by Student‘s t-test (**p<0.01). B) Therapeutic

vaccination protocol. 105 CT26-HA tumor cells were s.c. transplanted into the right

flank. Mice were immunized s.c. either with PBS, soluble CpG and HA peptides or

CpG and HA-peptide functionalized CaP nanoparticles at day 3, 5 and 7 post tumor

cell transplantation. C) Tumor volume was measured at indicated time points. The

results of 4 independent experiments are shown. n = 15. Statistical analysis was

performed by Two-Way ANOVA followed by Tukey‘s multiple comparisons test

(*p<0.05, **p<0.01, ****p<0.0001).


nanoparticles enhances the cytotoxic CD8+ T cell response. Lymphocytes were

isolated from the tumor-draining lymph node of immunized mice at day 12 post tumor

cell transplantation. A) Frequencies of CD8+, CD4+ or Foxp3 expressing CD4+ T

cells were determined by flow cytometry. B) Frequencies of Ki67 expressing CD8+ T

cells, GzmB expressing CD43+ CD8+ T cells or EOMES expressing CD8+ T cells.

Results of two independent experiments are depicted. Statistical analysis was

performed by one-way ANOVA followed by Tukey‘s multiple comparisons test

(*p<0.05, **p<0.01, ***p<0.001).


nanoparticles enhances the infiltration of the tumor by CD8+ T cells. Lymphocytes

were isolated from the tumor of vaccinated mice at day 12 post tumor cell

transplantation. A) Frequencies of CD8+, CD4+ and Foxp3+ T cells were determined

by flow cytometry. B) Frequencies of Ki67+ CD8+ T cells, CD43+ GzmB and EOMES

expressing CD8+ T cells. C) Frequencies of Klrg1+ CD8+ T cells and CD69+ CD8+ T

cells. D) Frequencies of TIM-3+ CD8+ T cells and PD-1+ CD8+ T cells. E) Frequencies

of CCR4+ CD8+ T cells, CCR5+ CD8+ T cells and CXCR3+ CD8+ T cells. F)

Correlation of the percentage of CD43+ GzmB expressing CD8+ T cells to the tumor




21

volume at day 12. Results of two independent experiments are depicted. G) Tumor

volume was measured at indicated time points. In some groups CD8+ T cells were

depleted by i.p. injection of αCD8 monoclonal antibody at day 3, 5 and 7. Results of

two independent experiments are depicted. Statistical analysis was performed by

one-way ANOVA followed by Tukey‘s multiple comparisons test. (*p<0.05, **p<0.01,

***p<0.001).

Figure 4: Enhanced CD8+ T cell immunity is dependent on IFN I induction. A)

Immunization protocol. 105 CT26-HA tumor cells were s.c. transplanted into the right

flank. Mice were immunized s.c. either with PBS or CpG and HA-peptide

functionalized CaP nanoparticles at day 3, 5 and 10 post tumor cell transplantation.

Additionally, mice were treated i.p. with αIFNAR monoclonal antibody at day 2 and 4.

B) Tumor volume was measured at indicated time points. The results of 2

independent experiments are shown. n = 8. Statistical analysis was performed by

Two-Way ANOVA followed by Tukey‘s multiple comparisons test (**p<0.01,

****p<0.0001). C-D) Frequencies of Ki67+ or GzmB+ CD43+ CD8+ T cells in the tumor

draining lymph node (C) or tumor (D) are shown. Results of two independent

experiments are depicted. Statistical analysis was performed by one-way ANOVA

followed by Tukey‘s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001).

Figure 5: Combination immunotherapy enhances CD8+ T cell immunity. A)

Immunization protocol. 105 CT26-HA tumor cells were s.c. transplanted into the right

flank. Mice were immunized s.c. either with PBS or CpG and HA-peptide

functionalized CaP nanoparticles at day 3, 5 and 7 post tumor cell transplantation.

Additionally, mice were treated i.p. with αPD-L1 monoclonal antibody at day 3 and 6.

B) Tumor volume was measured at indicated time points. The results of 3

independent experiments are shown. n = 10. Statistical analysis was performed by

Two-Way ANOVA followed by Tukey‘s multiple comparisons test (*p<0.05,

***p<0.001, ****p<0.0001). C-D) Frequencies of CD8+ T cells as well as Ki67+ or

GzmB+ CD43+ CD8+ T cells in the tumor draining lymph node (C) or tumor (D) are

shown. Results of 2 independent experiments are depicted. Statistical analysis was

performed by one-way ANOVA followed by Tukey‘s multiple comparisons test

(*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).




22

Figure 6: Functionalization of CaP nanoparticles with a primary tumor cell lysate enhances

the CD8+ T cell response and represses tumor growth. 105 CT26 tumor cells were s.c.

transplanted into the right flank. Mice were immunized s.c. either with PBS, soluble CpG and

tumor lysate, CpG and tumor lysate functionalized CaP nanoparticles, soluble CpG and HA

peptides or CpG and HA peptide functionalized CaP nanoparticles at day 3, 5 and 7 post

tumor cell injection. A) Tumor volume was measured at indicated time points. The results of

2 independent experiments are shown. n = 8. Statistical analysis was performed by Two-Way

ANOVA followed by Tukey‘s multiple comparisons test (**p<0.01, ****p<0.0001). B-C)

Frequencies of Ki67+ or GzmB+ CD43+ CD8+ T cells in the tumor draining lymph node (B) or

tumor (C) were determined by flow cytometry. Results of two independent experiments are

depicted. Statistical analysis was performed by one-way ANOVA followed by Tukey‘s

multiple comparisons test (*p<0.05, **p<0.01).




Published OnlineFirst April 8, 2019.Mol Cancer Ther Carolin Heße, Sebastian Kollenda, Olga Rotan, et al. tumor growth control in anti-tumor immunotherapyA tumor-peptide based nanoparticle vaccine elicits efficient

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