GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutics

Laboratory of General Biochemistry and Physical Pharmacy

Academic year 2014-2015

Dana DE SAEGHER

Master of Science in Industrial Pharmacy

Promoter

Prof. Dr. S. De Smedt

Commissioners

Prof. Dr. D. Deforce

Prof Dr. R. Kemel

Prof. Dr. H. Nelis

Prof. Dr. I. De Meester

Prof. Dr. P. Declerck

Prof. Dr. T. Vanhaecke

Dendritic cell-based cancer immunotherapy:

rational design of nanoparticle vaccines.

COPYRIGHT

"The author and the promoter give the authorization to consult and to copy parts of this

thesis for personal use only. Any other use is limited by the laws of copyright, especially

concerning the obligation to refer to the source whenever results from this thesis are cited."

January 7, 2015

Promoter Author

Prof. Dr. S. De Smedt Dana De Saegher

SUMMARY

Dendritic cells (DCs) form the bridge between the innate and adaptive immune

system. This makes them particularly interesting candidates to be used in cancer

immunotherapy strategies. In recent years, several dendritic cell-based cancer vaccination

strategies have emerged and proven their potential. However, the majority of DC-based

vaccines currently tested consist of antigen-loaded and stimulated autologous DCs, the so

called ex vivo DC vaccines. Despite their promise, economic, logistic and therapeutic

complexities limit the applicability of these ex vivo vaccines. Therefore, the development of

nanoparticle vaccines which could allow direct in vivo modification of DCs will be

advantageous.

In this study, we aimed to design a serum-stable lipid-based delivery tool that

stimulates DCs in vitro to induce potent cytotoxic T cell responses against specific tumor

associated antigens (TAAs). In order to stimulate DCs to induce potent antitumor cytotoxic T

cell responses, the lipid-based delivery tool has to (a) efficiently deliver mRNA encoded TAAs

into DCs, especially in the presence of serum and (b) stimulate full maturation of the TAA-

loaded DCs. In this study, we have demonstrated that, of all particles tested, DOTAP:CHOL

mRNA-lipoplexes (mRNA-LPXs) are the most efficient particulate systems to introduce mRNA

encoded proteins into BM-DCs in serum-containing medium. Furthermore, we showed that

DOTAP:CHOL mRNA-LPXs as such induce only limited DC maturation and that the

susceptibility to maturation stimuli was not negatively influenced by particle loading. In an

attempt to further increase the DC maturation-status, we included adjuvants (i.e. MPLA, CpG

ODN or TriMix mRNA) into the DOTAP:CHOL mRNA-LPXs. Our results show that co-

incorporation of all the above mentioned adjuvants enhanced DC maturation. With regard to

an enhanced expression of maturation markers, both MPLA and CpG ODN were more potent

than TriMix mRNA. Moreover, increased cytokine secretions were observed upon co-

incorporation of MPLA or CpG ODN while no increase was observed when TriMix mRNA was

included. Importantly, we proved that the MPLA and CpG ODN containing DOTAP:CHOL

mRNA-LPXs remained their capacity to deliver mRNA encoded proteins into BM-DCs.

However, with regard to TriMix mRNA, an expected decrease in transfection efficiency was

observed.

Ultimately, we investigated the capacity of particle-loaded DCs to stimulate antigen-

specific T cell proliferation and cytotoxic T lymphocyte (CTL) responses in vitro. Although we

could not yet prove significant differences in T cell proliferation for unmodified and

adjuvant-containing DOTAP:CHOL mRNA-LPXs, we observed an increase in antigen-specific

CTL responses upon co-incorporation of adjuvants into the DOTAP:CHOL mRNA-LPXs.

In conclusion, we were able to design a serum-stable lipid-based delivery tool that

stimulates DCs in vitro to induce potent cytotoxic T cell responses against specific TAAs. The

development of such a tool is a major step forward towards developing in vivo applicable

DC-vaccines. However, we must point out the need to perform in vivo research in order to

evaluate the clinical applicability of the designed particles.

SAMENVATTING

Dendritische cellen (DCs) vormen de brug tussen het aangeboren en het verworven

immuunsysteem. Dit maakt hen zeer geschikte kandidaten om te gebruiken in kanker

immunotherapie. De voorbije jaren is het aantal DC gebaseerde strategieën dan ook sterk

toegenomen. Bovendien hebben verschillende strategieën reeds hun potentieel bewezen.

Huidige klinische studies maken echter gebruik van de ex vivo aanpak waarbij ex vivo

gegenereerde DCs beladen worden met antigeen en na activatie opnieuw worden

geïnjecteerd. Ondanks de veelbelovendheid van deze aanpak, wordt de klinische

toepasbaarheid ervan belemmerd door economische, logistieke en therapeutische

moeilijkheden. De ontwikkeling van nanopartikel vaccinaties, die directe in vivo DC-

modificatie toelaten, zou dan ook zeer voordelig zijn.

In deze studie beoogden we een serum-stabiel nanopartikel te ontwikkelen dat in

staat is DCs in vitro te stimuleren om cytotoxische T cel (CTL) responsen op te wekken tegen

specifieke tumor geassocieerde antigenen (TAAs). Om dit mogelijk te maken, moet het

nanopartikel in staat zijn om (a) doeltreffend DCs in vitro te transfecteren met TAA coderend

mRNA en (b) volledige DC maturatie te induceren. In deze studie toonden we aan dat, van

alle geteste partikels, DOTAP:CHOL mRNA-lipoplexen (mRNA-LPXs) het meest doeltreffend

zijn om DCs te transfecteren met proteïne coderend mRNA in serum bevattend medium.

Bovendien werd er bewezen dat deze partikels slechts een beperkte invloed hebben op de

maturatie status en dat DCs ontvankelijk blijven voor maturatie stimuli. In een poging om de

DC maturatie status verder te verhogen werden adjuvantia (MPLA, CpG ODN of TriMix

mRNA) geïncorporeerd in de DOTAP:CHOL mRNA-LPXs. We toonden aan dat co-incorporatie

van al deze adjuvantia een verhoging in DC maturatie status veroorzaakte. Met betrekking

tot een verhoogde expressie van maturatie merkers waren zowel MPLA als CpG ODN

potenter dan TriMix mRNA. Bovendien werden verhoogde cytokine secreties waargenomen

wanneer DCs beladen werden met MPLA en CpG ODN bevattende mRNA-LPXs terwijl dit niet

geval was bij belading met TriMix bevattende mRNA-LPXs. Hoewel bij TriMix mRNA een

verwachte daling in transfectie efficiëntie waargenomen werd, was dit niet het geval

wanneer MPLA en CpG ODN werden geïncorporeerd in de mRNA-LPXs.

Tot slot werd nagegaan in welke mate partikel-beladen DCs in staat zijn om in vitro

antigeen-specifieke T cel proliferatie en CTL responsen te induceren. Ondanks het feit dat we

nog geen significant verschil in T cel proliferatie tussen klassieke en adjuvantia-bevattende

DOTAP:CHOL mRNA-LPXs konden aantonen, konden we toch een stijging in antigeen-

specifieke CTL responsen waarnemen bij co-incorporatie van adjuvantia in de mRNA-LPXs.

In dit project waren we in staat een serum-stabiel nanopartikel te ontwikkelen dat

dat in staat is DCs in vitro te stimuleren om CTL responsen op te wekken tegen specifieke

TAAs. Dit is een enorme vooruitgang in de ontwikkeling van in vivo DC vaccinaties. Verder in

vivo onderzoek is echter noodzakelijk om de klinische toepasbaarheid van de ontworpen

partikels te evalueren.

DANKWOORD

In de eerste plaats wil ik graag mijn promotor, Prof. Dr. S. De Smedt bedanken om mij de

mogelijkheid te bieden om deze masterproef uit te voeren binnen zijn onderzoeksgroep en

om mij toe te laten de professionele apparatuur in het laboratorium te gebruiken voor het

uitvoeren van mijn experimenten.

Allermeest zou ik graag mijn begeleider Rein Verbeke willen bedanken. Gedurende de hele

thesisperiode kon ik steeds rekenen op je advies en steun bij het experimentele werk. Vol

enthousiasme stond je steeds klaar om uitleg te geven, mee te helpen met de experimenten

en oplossingen te zoeken waar nodig. Bedankt voor het nalezen en zorgvuldig verbeteren van

deze masterproef. Zonder jou hulp zou ik nooit hetzelfde resultaat bereikt hebben. En tot slot

ook bedankt voor de kans die ik kreeg om in vivo experimenten mee te volgen.

Graag zou ik ook Heleen Dewitte bedanken voor de zorgvuldige uitleg en hulp bij nieuwe

experimenten. Ook bedankt om de in vivo experimenten mogelijk te maken.

Tevens wil ik ook nog alle professoren, doctoraatstudenten en postdocs bedanken voor de

vragen die ik hen mocht stellen.

Mijn dank gaat ook uit naar mijn mede-thesis-studenten voor de aangename werksfeer, de

grappige momenten en de aangename ontspanning tijdens de pauzes. Zonder jullie zou het

nooit hetzelfde geweest zijn.

Als laatste wens ik graag mijn familie en vrienden te bedanken voor de steun en interesse die

jullie toonden tijdens deze thesis.

CONTENTS

1. INTRODUCTION ............................................................................................................. 1

1.1. CANCER ............................................................................................................................ 1

1.1.1. A key public health concern? ............................................................................ 1

1.1.2. Pathology ........................................................................................................ 1

1.1.3. Treatments ...................................................................................................... 2

1.1.3.1. Conventional treatments .................................................................................. 2

1.1.3.2. New and arising therapies ................................................................................. 3

1.2. THE IMMUNE SYSTEM: A TOOL TO COMBAT CANCER? .................................................. 4

1.2.1. Tumor immune surveillance ............................................................................. 4

1.2.1.1. Tumor-associated antigens ............................................................................... 4

1.2.1.2. Dendritic cells: initiators of the cellular immune response .............................. 4

1.2.1.3. Antitumor effector cells .................................................................................... 6

1.2.2. Cancer immunotherapy .................................................................................... 7

1.2.2.1. Adoptive T cell therapy ..................................................................................... 7

1.2.2.2. Antibody therapy............................................................................................... 8

1.2.2.3. DC vaccination ................................................................................................... 9

1.3. IN VIVO DC VACCINATION ............................................................................................. 11

1.4. MRNA-LIPOPLEXES: THE BASICS .................................................................................... 12

2. OBJECTIVES ................................................................................................................. 15

3. MATERIALS AND METHODS ......................................................................................... 17

3.1. MRNA PREPARATION .................................................................................................... 17

3.1.1. Green fluorescent protein (GFP) mRNA .......................................................... 17

3.1.2. Ovalbumin (OVA) mRNA ................................................................................ 18

3.2. LIPOPLEX PREPARATION ................................................................................................ 18

3.2.1. Unmodified mRNA-LPXs ................................................................................. 18

3.2.1.1. Lipid stock solutions ........................................................................................ 18

3.2.1.2. Liposome preparation ..................................................................................... 19

3.2.1.3. mRNA loading of the liposomes ...................................................................... 19

3.2.2. Immunomodulatory DOTAP:CHOL mRNA-LPXs ............................................... 20

3.3. LIPOPLEX CHARACTERIZATION ...................................................................................... 20

3.3.1. Dynamic light scattering (DLS) ........................................................................ 21

3.3.2. Zeta potential measurements ........................................................................ 21

3.4. MRNA COMPLEXATION ................................................................................................. 21

3.5. CELL CULTURE ................................................................................................................ 22

3.6. LIPOPLEX LOADING OF DCS ........................................................................................... 23

3.6.1. Loading in culture medium (5% FCI serum) ..................................................... 23

3.6.2. Loading in serum reduced medium (Opti-MEM®) ........................................... 23

3.7. TRANSFECTION EFFICIENCY AND MATURATION STATUS ............................................. 23

3.8. IN VITRO T CELL PROLIFERATION ASSAY ....................................................................... 24

3.9. IN VITRO CYTOTOXIC T LYMPHOCYTE (CTL) ASSAY ....................................................... 25

3.10. CYTOKINE SECRETION ................................................................................................. 26

3.11. STATISTICAL ANALYSIS................................................................................................. 26

4. RESULTS ...................................................................................................................... 27

4.1. PARTICLE CHARACTERIZATION ...................................................................................... 27

4.2. TRANSFECTION EFFICIENCY ........................................................................................... 30

4.3. DENDRITIC CELL ACTIVATION ........................................................................................ 31

4.3.1. DC activation by unmodified DOTAP:CHOL mRNA-LPXs .................................. 31

4.3.2. DC activation by immunomodulatory DOTAP:CHOL mRNA-LPXs ..................... 32

4.3.3. Influence on transfection ............................................................................... 36

4.4. INDUCTION OF CD8+ T CELL PROLIFERATION ............................................................... 37

4.5. INDUCTION OF ANTIGEN SPECIFIC CYTOTOXIC T CELL (CTL) RESPONSES .................... 38

5. DISCUSSION ................................................................................................................ 40

6. CONCLUSION .............................................................................................................. 46

7. REFERENCES ................................................................................................................ 48

LIST OF ABBREVIATIONS

APC Antigen presenting cell

APC Allophycocyanin

BM-DC Bone marrow-derived DC

BSA Bovine Serum Albumine

CAR Chimeric antigen receptor

CCR7 Chemokine (C-C) receptor 7

CD Cluster of differentiation

CFSE Carboxyfluorescein succinimidyl ester

CPD Cell proliferation dye eFluor 670

CpG ODN CpG oligodeoxynucleotides

CTL Cytotoxic T lymphocyte

CTLA-4 Cytotoxic T-lymphocyte antigen 4

DAMP Danger-associated molecular pattern

DC Dendritic cell

DC-chol 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol

DLS Dynamic light scattering

DMSO Dimethyl sulphoxide

DNA Deoxyribonucleic acid

DOPE Dioleoylphosphatidylethanolamine

DOTAP 1,2-dioleoyl-3-trimethylammonium-propane

DPBS Dulbecco's Phosphate-Buffered Saline

ELISA Enzyme-linked immunosorbent assay

FBS Fetal bovine serum

FCI serum FetalCloneTM I serum

FDA Food and Drug Administration

FSC Forward scattered light

GFP Green fluorescent protein

GM-CSF Granulocyte macrophage colony-stimulating factor

HRP Horseradish peroxidase

iDC Immature DC

IL Interleukin

LPS Lipopolysaccharide

LPX Lipoplex

MHC Major histocompatibility complex

MoDC Monocyte-derived dendritic cell

MPLA Monophosphoryl lipid A

mRNA Messenger ribonucleic acid

N/P ratio Lipid-to-mRNA charge ratio

NK cell Natural killer cell

OVA Ovalbumin

PAMP Pathogen-associated molecular pattern

PBMC Peripheral blood monocyte

PD-1 Programmed cell death protein 1

pDNA Plasmid DNA

PE Phycoerythrin

PRR Pathogen recognition receptor

RNAiMAX Lipofectamine® RNAiMAX

SPF Specific-pathogen-free

SSC Side scattered light

TAA Tumor-associated antigen

TCR T cell receptor

TE Transfection efficiency

TH cell T helper cell

TIL Tumor infiltrating lymphocyte

TLR Toll-like receptor

TMB Tetramethylbenzidine

TNFR Tumor necrosis factor receptor

1

1. INTRODUCTION

1.1. CANCER

1.1.1. A key public health concern?

Cancer is one of the leading causes of mortality in Europe. In fact, in 10 European

countries, including Belgium, cancer has surpassed cardiovascular disease as the main cause

of death among men. Moreover, in Denmark, the same was observed among woman for the

first time. (Nichols et al., 2014) In Europe, according to the GLOBOCAN 2012 project, 3.8

million new cases of cancer were diagnosed, causing 1.9 million deaths. The four most

common types of cancer include breast, prostate, lung and colorectal cancer with lung

cancer showing the highest mortality. (Ferlay et al., 2014) Furthermore, in 2009, the

estimated economic burden of cancer in Europe was 126 billion euro, with health care and

productivity losses due to early death being the major costs accounting for 51 billion euro

and 42,6 billion euro respectively. These statistics demonstrate that cancer is a key public

health concern with a high impact on the society. (Luengo-Fernandez et al., 2013)

1.1.2. Pathology

Cancer is a disease that involves uncontrolled cell proliferation and disrupted cell

homeostasis, as a result of regulation failure caused by multiple genetic or epigenetic

alterations in the genome of a normal cell. These alterations in genes can either occur at

random, be inherited, or may be caused by exposure to carcinogens (e.g. tobacco, chemicals,

radiation and infectious agents). (Macaluso et al., 2003; Sadikovic et al., 2008) In virtually all

types of cancer cells, six crucial alterations in cell physiology were observed. These so called

cancer hallmarks, as described by Hanahan and Weinberg, include (a) self-sufficiency in

growth signals, (b) insensitivity to growth-suppressor signals, (c) resistance to apoptosis, (d)

replicative immortality, (e) inducing and sustaining angiogenesis and (f) tissue invasion and

spreading (i.e. metastasis). (figure 1.1.) Although, during their development, all cancer cells

acquire this same set of hallmark capacities, their mode of doing so varies mechanistically

and chronologically. (Hanahan and Weinberg, 2011, 2000)

2

As mentioned in the previous paragraph, oncogenic transformed cells are able to

move out of the primary tumor mass, invade adjacent tissues and hence spread to other

organs were they found new settlements of tumor cells, the so called metastases. By

impairing tissue’s functionality or by causing an increased pressure on the surrounding,

primary tumors and metastases can give rise to a wide range of symptoms which depend on

different tumor characteristics such as its location and extensiveness. When untreated,

invasion and metastasis will cause death due to a fatal decrease in vital organ functionality.

(Hanahan and Weinberg, 2000)

Figure 1.1. The Hallmarks of Cancer. Six essential alterations observed in the cell physiology

of a cancer cell. (Hanahan and Weinberg, 2011)

1.1.3. Treatments

1.1.3.1. Conventional treatments

To date, two of the most routinely applied therapies for the treatment of cancer are

radiotherapy and surgery. Although these local physical methods show some side effects

such as postoperative pain and healthy tissue damage respectively, they have proven to be

successful, especially for the eradication of primary localized tumors. However, these

strategies show limitations in treatment of inaccessible tumor places, non-solid tumors

(e.g. leukemia) and disease relapse due to metastases. (Urruticoechea et al., 2010)

Therefore, chemotherapy is an often required cancer therapy. This systemic

chemical method targets fast proliferating cells, such as tumor cells, by interfering with the

cell division process and the cellular DNA (e.g. alkylating agents, antimetabolites, anti-

microtubule agents, antitumor antibiotics and topoisomerase inhibitors). Unfortunately

chemotherapeutics can be rendered ineffective by the occurrence of cancer cell resistance.

3

Chemotherapeutics also have the disadvantage of indiscriminately targeting all rapidly

dividing cells. Thereby, chemotherapy also harms healthy cells, mainly those of the gastric

and hematopoietic system, causing many side effects (e.g. nausea, anemia, hair loss).

(Urruticoechea et al., 2010; Vanneman and Dranoff, 2012; Wayteck et al., 2014)

1.1.3.2. New and arising therapies

With the aim of decreasing troubling systemic side effects, new approaches that

are more selective to tumor cells have emerged. One of these new strategies is the use of

nanoparticle carriers to more specifically target chemotherapeutic drugs directly to tumor

cells. (Kateb et al., 2011; Krishnamachari et al., 2011) For example, Doxil®, a liposomal

doxorubicin formulation, is the first nano-drug which was approved by the FDA (1995).

(Barenholz, 2012)

More recently, a better insight in tumor pathogenesis and tumor immune

surveillance boosted the development of these new selective therapies even more by

providing new treatment options, including tumor-targeted therapies and cancer

immunotherapy. Targeted therapies act by inhibiting essential biological pathways that are

crucial for tumor growth and development. Most targeted therapies are either monoclonal

antibodies or small molecule inhibitors with distinct mechanisms of action such as blockage

of growth factor receptors, angiogenesis inhibition and apoptosis induction. Although

multiple targeted therapies show tumor regression, their clinical benefit is short-lived due

to an acquired cancer cell resistance. (Vanneman and Dranoff, 2012; Wayteck et al., 2014)

Besides targeted therapies, recent successes in cancer immunotherapy have

validated the value of this novel therapeutic approach in the combat of cancer. The aim of

immunotherapy is to stimulate a patient’s own immune system to destruct the tumor.

Besides its tumor selective nature, main advantages of this strategy are (a) that it can be

universally applied to treat different types of cancer and (b) that it provides a durable

protection through the generation of immune memory. (Vanneman and Dranoff, 2012) In

the next chapter, we will briefly discuss the basic immunological mechanisms after which we

will focus on the main cancer immunotherapy breakthroughs.

4

1.2. THE IMMUNE SYSTEM: A TOOL TO COMBAT CANCER?

1.2.1. Tumor immune surveillance

Different breakthroughs during the years have improved our knowledge of the

complex function of the immune system. It became clear that, besides providing protection

against infectious agents, the immune system can also detect and eliminate continuously

arising, transformed cells.

1.2.1.1. Tumor-associated antigens

A first breakthrough was the identification of antigens selectively, preferentially or in

excess expressed by tumor cells. These non-self-proteins are able to trigger the immune

system and are known as tumor-associated antigens (TAAs). For long, the existence of TAAs

was only presumed. However, in the 1940s and 1950s, experiments by Gross, Foley and

Prehn showed that tumors were antigenic when implanted in mice (Foley, 1953; Gross,

1943; Prehn and Main, 1957). As early as 1965, the first evidence of the existence of TAAs

was provided by Gold and Freedman, which was later confirmed by Parker and Rosenberg

(Gold and Freedman, 1965; Parker and Rosenberg, 1977). (Dewitte et al., 2014b; Wayteck et

al., 2014) To date, several TAAs are already identified and generally classified into two main

classes: tumor-specific antigens, only present on tumor cells, and self-antigens, expressed on

both tumor cells and normal cells. The class of tumor-specific antigens encompasses

mutation-derived antigens and viral antigens. Self-antigens can be subdivided into

overexpressed antigens, tissue-differentiation antigens and cancer testis antigens. (Wayteck

et al., 2014)

1.2.1.2. Dendritic cells: initiators of the cellular immune response

Another crucial breakthrough was the identification of dendritic cells (DCs). In 1973,

Ralph Steinman first described DCs as the messengers between the two functional

subsystems of the immune system, the innate and the adaptive immunity. For this discovery,

he was rewarded with the Nobel Prize in Physiology or Medicine in 2011. More specific, DCs

capture antigens in peripheral tissues through several endocytic pathways. While migrating

to the secondary lymphoid tissues (e.g. lymph nodes, spleen), DCs process those captured

antigens into peptides and present them on their surfaces in association with major

5

histocompatibility complexes (MHCs) class I or class II. (Banchereau and Steinman, 1998;

Dewitte et al., 2014b; Palucka and Banchereau, 2012)

Generally, antigens presented by MHC class I molecules are derived from intracellular

generated cytoplasmic proteins. First, these proteins are degraded to peptide fragments by

the proteasome. The generated peptides are then transferred to the endoplasmic reticulum

where they bind to MHC class I molecules. Subsequently, the loaded class I MHCs are

exported for binding at the surface of the cell. Contrarily, antigens presented by MHC class II

molecules are derived from engulfed extracellular proteins. After uptake by endocytosis,

proteases within the formed vesicles degrade the proteins into peptides. Subsequently,

peptide containing vesicles fuse with MHC class II containing vesicles after which the

constituted MHC II/peptide complexes are transported for presentation on the cell surface.

(figure 1.2.) However, in contrast to other antigen presenting cells (APCs), DCs are the only

cells that are capable of presenting peptides derived from exogenous proteins in MHC class I

molecules through a process called cross-presentation. This enables DCs to engulf

extracellular TAAs derived from dying tumor cells and present them in both class I and class

II MHC molecules. (Banchereau and Steinman, 1998; Steinman and Banchereau, 2007)

Figure 1.2. Antigen presentation by the MHC class II and class I pathway (Abbas, 2010)

6

In the secondary lymphoid tissues (e.g. lymph nodes, spleen), DCs encounter T

lymphocytes, all with a unique antigen specific T cell receptor (TCR). Through this event, DCs

can activate the encountered T cells, which requires 3 signals. The first signal is a highly

specific interaction between the TCR of naïve CD4+ T cells or CD8+ T cells and the MHC

II/antigen or MHC I/antigen complex present on the DC surface respectively. The second

signal is the interaction between co-stimulatory molecules, most notably CD80 and CD86, on

the DC surface and their corresponding receptor (i.e. CD28) on the specific T cell surface.

Lastly, secretion of immunostimulatory cytokines (e.g. IL-12) provides the third signal for

activation. (Benencia et al., 2012; Benteyn et al., 2014; Vanneman and Dranoff, 2012)

Figure 1.3. Three signals required for T cell activation (Pollard et al., 2013)

1.2.1.3. Antitumor effector cells

When activated, naïve T cells will proliferate and differentiate into effector T cells

with the same receptor specificity. Naïve CD8+ T cells will proliferate and differentiate into

effector cytotoxic T cells (CTLs) which can induce apoptosis of tumor cells presenting TAAs in

MHC I molecules. This can occur either directly, trough the release of perforin and

granzymes or by activating the Fas/Fas ligand pathway, or indirectly, by releasing tumor

necrosis factor-α (TNF-α) and interferon-ƴ (IFN-ƴ). In contrast, naïve CD4+ T cells will

proliferate and differentiate into effector T helper cells (TH cells). Emerging evidence suggest

that T helper cells promote the differentiation of naïve CD8+ T cells into cytotoxic T cells

(CTLs). Moreover, studies also show that TH cells play an important role in regulating the

generation and persistence of long-term memory CD8+ T-cells which are needed to generate

an accelerated immune response when the same antigen is re-encountered. (Andersen et

al., 2006; Knutson and Disis, 2005; Silva et al., 2013)

7

Furthermore, DCs are not only capable of inducing T cell responses. They also

communicate with natural killer cells (NK cells), as they are able to stimulate NK cells by

soluble (e.g. IL-12, IL-15) and contact-signals (e.g. NKp30). NK cells possess the unique

capacity to identify and destroy cells that express decreased levels of MHC class I molecules.

Under normal circumstances, these MHC class I molecules are ubiquitously expressed on

virtually all autologous cells (i.e. normal and cancerous cells). However, selective pressure

can give rise to MHC I deficient tumor cells. These MHC I deficient tumor cells are no longer

recognized by CTLs but become vulnerable for NK cell cytotoxic activities (e.g. via cytotoxic

granules). Furthermore, besides a direct effect, NK cells also indirectly stimulate the

elimination of tumor cells by facilitating the activation, maturation and cytokine production

of DCs. (Lion et al., 2012; Purdy and Campbell, 2009; Smyth et al., 2005)

1.2.2. Cancer immunotherapy

Unfortunately, tumor cells are still able to escape immunity through a variety of

mechanisms. In recent years, increasing evidence has highlighted the importance of the

tumor microenvironment. This supporting environment harbors a variety of cell types which

inhibit antitumor immune responses through several mechanisms (e.g. blocking the activity

of antitumor effector cells) thereby enabling tumor progression. (Dewitte et al., 2014b)

By stimulating the patient’s own immune system, immunotherapy aims to shift the

system back into balance. Several strategies have been devised so far, ranging from (a)

strategies aimed at providing or supporting TAA-specific T cells, such as adoptive T cell

therapy or antibody therapy to (b) strategies aimed at inducing DC initiated anti-tumor

immune responses, namely DC vaccination. In the next paragraphs, we will shortly introduce

the basics of adoptive T cell therapy and antibody therapy, two therapies that have been

recently awarded with the title ‘Science breakthrough of the year 2013’. Subsequently, we

will focus on DC vaccination, the focal point of our research.

1.2.2.1. Adoptive T cell therapy

ACT involves the transfer of in vitro selected and multiplied tumor specific reactive T

cells into the patient. This can be achieved by isolating T lymphocytes from (a) either the

tumor itself, in case of tumor infiltration by anti-tumor T cells, or (b) the peripheral blood.

Using the first method, T cells isolated from the resected tumor mass (i.e. tumor infiltrating

8

lymphocytes (TILs)) which show tumor reactivity are selected. Secondly, the tumor-reactive

TILs are stimulated to multiply after which they can be re-infused. Alternatively, T cells

isolated from the patient’s blood can be engineered to recognize certain tumor antigens by

transducing them with viral vectors encoding an antigen-specific T cell receptor or a

chimeric antigen receptor (CAR). (Wayteck et al., 2014)

During a study performing the first technique on 93 metastatic melanoma patients,

complete remission was induced in 22% of the patients with long-termed effects in 19 out

of 20 patients. The second technique was tested during a clinical trial treating patients with

metastatic synovial cell sarcoma and melanoma using a viral vector encoding TCRs towards

NY-ESO-1 antigens. Complete remission that persisted over a year was induced in 2 out of

11 patients. (Wayteck et al., 2014)

1.2.2.2. Antibody therapy

As mentioned above, three signals are needed to activate naïve T-cells: (1)

recognition of the MHC-antigen complexes by TCRs, (2) additional co-stimulation signals and

(3) cytokine stimulation. Co-stimulatory signals are provided through a variety of

transmembrane proteins of the B7 and tumor necrosis factor receptor (TNFR) family (e.g. 4-

1BB). Contrarily, T cells also receive co-inhibitory signals, known as immune checkpoints,

through interactions between co-inhibitory receptors, such as cytotoxic T-lymphocyte

antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) and their ligands,

CD80(B7.1)/CD86(B7.2) and PD-L1/2 (B7H1/B7DC) respectively. Those negative regulatory

signals are able to decrease T cell activation or induce immune tolerance. One of the

strategies devised to enhance T cell function is the blockage of those co-inhibitory signals

through the use of monoclonal antibodies that block the co-inhibitory receptors CTLA-4 and

PD-1. (Vanneman and Dranoff, 2012)

Ipilimumab, an anti CTLA-4 treatment, is already approved by the FDA as a treatment

of metastatic melanoma while PD-1 blocking antibodies are extensively researched. For

example, an anti-PD-1 antibody tested in a clinical trial is Nivolumab, which shows as a

promising treatment for patients with metastatic non-small cell lung carcinoma, melanoma

and renal cell carcinoma. (Wayteck et al., 2014)

9

1.2.2.3. DC vaccination

DCs are of major importance in driving potent anti-tumor immune responses as

they are capable of initiating the activity of different arms of the immune system, including

T cells. Therefore, the use of DC-based strategies to induce immunological tumor

regression is extensively investigated. (Steinman and Banchereau, 2007)

The majority of DC-based vaccines currently tested in clinical trials consist of mature

antigen-loaded autologous DCs and are called ex vivo DC vaccines. Generating those

vaccines, different steps can be distinguished. First, patient’s DC precursor cells (i.e.

peripheral blood monocytes (PBMCs)) are isolated and differentiated into DCs (i.e.

monocyte-derived DCs (MoDCs)) through the use of cytokines (e.g. granulocyte

macrophage colony-stimulating factor (GM-CSF), interleukine 4 (IL-4)). As mentioned in

previous sections, in order to induce the initiation of cellular immunity, DCs must provide

three signals: antigen presentation on MHCs, additional co-stimulation signals and cytokine

stimulation. In DC-based immunotherapy, DCs are modified to present TAAs and deliver

the 2nd and 3rd signal via 2 essential steps: (a) tumor-specific antigen loading and (b) DC-

maturation.(Dewitte et al., 2014b)

With regards to ex vivo DC-loading, different antigen preparations have already

been tested. DCs have been pulsed with tumor cells, tumor-derived proteins or peptides

and later on genetic antigen delivery approaches have been explored, such as TAA

encoding DNA or mRNA delivery. (Dewitte et al., 2014b)

After DC-loading, immature DCs (iDCs) are stimulated to mature. (Dewitte et al.,

2014b) This process is crucial due to the fact that, although immature DCs (iDCs) can

efficiently capture antigens through several endocytic pathways, iDCs lack co-stimulatory

signals which makes them unable to trigger naïve T-cell activation. Inadequate activation

will cause DCs to turn tolerogenic instead of immune stimulating, creating tumor immune

tolerance. (Banchereau and Steinman, 1998) Due to DC maturation a variety of changes are

initiated, including an (a) increased expression of co-stimulatory molecules (e.g. CD40,

CD80, CD86) and (b) production of pro-inflammatory cytokines (e.g. IL-12), earlier referred

to as the much needed 2nd and 3rd signal. Moreover, additional changes are observed

during maturation, such as (c) a downregulation of antigen-capture activity, (d) an

10

increased antigen presentation (i.e. increased expression of MHC class II) and (e) the

upregulated expression of chemokine receptors (e.g. CCR7), which allows DC-migration

into the lymph nodes. (Palucka and Banchereau, 2012) DC maturation can be obtained

through exposure to maturation stimuli. These stimuli can be pathogen-associated

molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) that trigger

intracellular or membrane bound pathogen recognition receptors (PRRs). Several factors

have been used to induce DC maturation. A well-known example is lipopolysaccharide (LPS)

which binds to the toll-like receptor (TLR) 4. Finally, the mature antigen-loaded DCs can be

re-injected into the patient.

Figure 1.4. Ex vivo DC vaccination

Clinical trials testing ex vivo DC-vaccines show promising results. Recently, Sipuleucel-

T (Provenge®), a treatment for asymptomatic or minimally symptomatic hormone

refractory prostate cancer, has been approved by the FDA. Clinical trials showed a

prolonged survival upon treatment with this ex vivo vaccine. (Wayteck et al., 2014)

11

Although the results are promising, a number of limitations hinder the applicability of

the current ex vivo DC-vaccines. First of all, ex vivo generation of DCs is very labor-

intensive, patient-specific and expensive due to the fact that all modification steps must be

performed on patient-specific isolated cells. Additionally, poor DC survival and migration to

the lymph nodes, where T cell activation takes place, is observed when DC-vaccines are

injected subcutaneously. Loss of activation and inadequate production of cytokines by the

injected cells are other important drawbacks of ex vivo DC-based vaccines. To overcome

these limitations, in vivo modification of DCs should be very beneficial. As a plus, using in

vivo DC vaccines, different kinds of DCs can be addressed creating a more robust immune

response. (Dewitte et al., 2014b; Phua et al., 2014; Tavernier et al., 2011)

1.3. IN VIVO DC VACCINATION

Even though, during in vivo DC vaccine generation, DC modification takes place in vivo

instead of ex vivo, the general principle of in vivo DC-vaccines is comparable to that of ex

vivo DC vaccines, still requiring the two essential modification steps: TAA loading and

maturation of DCs.

For in vivo DC antigen loading, researchers have moved on to the use of genetic

antigen delivery approaches, namely the use of TAA encoding DNA and mRNA. As compared

to plasmid DNA, mRNA does not require integration into the genome as it can directly be

translated in the cytoplasm. This results in favorable safety profiles (i.e. no insertional

mutagenesis), increased transfection of non-dividing cells such as DCs and fast but transient

expression. Moreover, due to efforts that enhanced the biological stability of mRNA and

increased its translation, mRNA based DC loading has emerged as a promising strategy.

(Phua et al., 2014; Tavernier et al., 2011)

The use of mRNA antigen delivery offers several other benefits. One of the main

advantages using mRNA is its flexibility, enabling the generation of different proteins of

interest. Secondly, encoding whole tumor antigen proteins, T cell responses against a wide

variety of epitopes can be activated. Thirdly, as the cytoplasmic protein production upon

mRNA delivery will mainly result in MHC class 1 presentation, induction of antitumor CTLs

will be favored. In addition, a prolonged duration of antigen presentation upon the use of

12

mRNA will result in an enhanced activation of those CTLs. Finally, mRNA is easy and cost

efficient to produce, delivering a high quality product. (Van Lint et al., 2014)

Although several clinical trials show an induction of potent immune responses through

the direct injection of naked mRNA into the lymph nodes, administration of naked mRNA via

subcutaneous or intravenous injections has failed the test. (Phua et al., 2014; Van Lint et al.,

2014) Probably due to (a) an inefficient uptake of the TAA encoding mRNA by DCs and/or (b)

rapid mRNA degradation by ribonucleases. To overcome these hurdles, the use of different

delivery systems, such as nanoparticles, is being increasingly investigated. Besides (a)

increasing the cellular uptake of mRNA and (b) providing protection against degradation,

additional advantages of nanocarriers include (c) a prolonged presentation of the antigen by

slowing down the mRNA-release (i.e. reservoir effect) and (d) the unique possibility to create

a multi-component system. (Krishnamachari et al., 2011; Pollard et al., 2013; Sahin et al.,

2014; Silva et al., 2013; Tavernier et al., 2011)

In this study we will focus on mRNA-lipoplexes (mRNA-LPXs), as a strategy for TAA-

encoding mRNA delivery in vivo. However, an in vivo applicable particle should not only

deliver the antigen, but also induce full maturation of the TAA-loaded DCs. To date, it

remains unclear whether LPXs as such are capable of inducing DC maturation. In fact, for

most particles, the co-incorporation of immune potentiating adjuvants (e.g. the TLR agonist

CpG oligodeoxynucleotides (ODN)) is favorable as it results in more effective in vivo DC

vaccines. (Remaut et al., 2007) In this way, it is optimal to generate a three component

system which contains (a) the TAA encoding mRNA, (b) an immune stimulating adjuvant and

(c) the particulate carrier itself. (Dewitte et al., 2014b)

1.4. mRNA-LIPOPLEXES: THE BASICS

mRNA-LPXs consist of cationic liposomes and mRNA. Cationic liposomes are composed

of phospholipids with an amphiphilic character, implying that they contain a hydrophilic

head region and a hydrophobic tail region. In an aqueous environment self-assembly of

dispersed phospholipids results in the formation of nanosized spherical vesicles consisting of

one or multiple lamellar phase lipid bilayers, so called unilamellar or multilamellar

liposomes. (Remaut et al., 2007)

13

For transfection application, cationic liposomes generally consist of cationic lipids and

neutral helper lipids. Cationic lipids possess a positively charged amine head group, which is

important for binding the negatively charged mRNA. Often used cationic lipids include 1,2-

dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N',N'-dimethylaminoethane)-

carbamoyl]cholesterol (DC-chol). Helper lipids are often used to facilitate endosomal escape

through their membrane destabilizing effects, as will be discussed later on. One of the most

commonly used helper lipids is dioleoylphosphatidylethanolamine (DOPE). (Balazs and

Godbey, 2011)

Mixing cationic liposomes with negatively charged mRNA, mRNA-LPXs are

spontaneously formed during a multistep process. First, electrostatic interactions arise

between the phosphate (mRNA) and the amine group (cationic lipids). The resulting defects

in the liposomal bilayer, triggers extensive lipid mixing, membrane merging and aggregate

growth. Eventually, proper mRNA packaging occurs in which mRNA is trapped within the

lipid-bilayers. (Remaut et al., 2007; Wasungu and Hoekstra, 2006) In a similar way, adjuvants

such as TriMix mRNA and CpG ODN can be co-incorporated in the lipid-based delivery

system.

With regard to cellular processing of nanoparticles (e.g. by DCs), different steps can be

distinguished, namely (a) cellular attachment, (b) internalization via endocytosis and (c)

intracellular trafficking. Cellular attachment of cationic nanoparticles is believed to be driven

by electrostatic interactions between the positively charged nanoparticles and negatively

charged molecules on the cell surface (e.g. proteoglycans). (Vercauteren et al., 2012) After

cellular attachment, nanoparticles are taken up by cells via endocytosis, which is the

generation of intracellular vesicles (i.e. endocytic vesicles) through the invagination of the

plasma membrane. Different endocytic pathways can be distinguished, including clathrin

dependent endocytosis, clathrin independent endocytosis, phagocytosis and

macropinocytosis. (Vercauteren et al., 2012; Wasungu and Hoekstra, 2006) To date, little is

known about the relative contribution of either pathway in the internalization of different

types of LPXs by different cell types. When the nanoparticles are internalized, intracellular

trafficking of the formed vesicles takes place. It is widely accepted that in many cases,

endocytic vesicles fuse with early/sorting endosomes. Subsequently, the early endosomes

can be transported (i.e. directly or indirectly) to the plasma membrane. Alternatively, early

14

endosomes can mature into late endosomes after which fusion with lysosomes can occur.

With regard to mRNA-LPXs, fusion with lysosomes will result in mRNA-LPXs degradation.

Thus, in order to be effective, the mRNA-LPXs have to be able to escape the endosomal

compartment. (De Haes et al., 2012; Vercauteren et al., 2012) Various strategies to induce

endosomal escape have already been devised so far. For example, endosomal escape can be

induced by cationic LPXs via close contact with the anionic lipids from the inner aspect of the

endosomal membrane. Formation of charge-neutralized ion pairs through diffusion of the

anionic lipids into the cationic LPXs subsequently results in endosomal membrane

destabilization. Moreover, the inclusion of DOPE into the lipoplexes also seems to facilitate

endosomal escape by promoting conversion of lipoplexes from a lamellar to a non-lamellar

hexagonal phase, which increases the chance of endosomal membrane destabilization and

mRNA release into the cytoplasm. (De Haes et al., 2012; Remaut et al., 2007; Wasungu and

Hoekstra, 2006) However, despite these efforts, endosomal release remains a major

obstacle.

15

2. OBJECTIVES

DC-based cancer vaccination has shown great potential in cancer immunotherapy.

The majority of DC-based vaccines currently tested in clinical trials consist of antigen-loaded

and stimulated autologous DCs, the so called ex vivo DC vaccines. Despite their promise,

these ex vivo DC vaccines are limited by economical, logistical and therapeutic complexities.

Therefore, developing more robust vaccines which modify DCs in vivo will be particularly

interesting. In this study, we aim to develop a lipid-based mRNA delivery tool that stimulates

DCs to induce potent antigen specific antitumor immune responses (i.e. cytotoxic T-cell

responses).

Firstly, we aim to develop and characterize a lipid-based mRNA delivery system that

is capable of efficiently introducing TAAs into murine bone marrow derived DCs in vitro,

especially in the presence of serum. Latter is of major importance since previous studies

show that several widely investigated cationic lipid-based mRNA delivery systems show a

high transfection efficiency in serum-free medium but lose their activity in the presence of

serum, making them not useful for in vivo DC vaccination applications. Therefore, the

development of a serum-stable delivery system, that could be used for in vivo applications is

our main challenge.

Besides efficiently introducing TAAs, the lipid-based mRNA delivery system has to

stimulate a full maturation of the TAA-loaded DCs in order to induce potent cytotoxic T-cell

responses. In a second phase of this study, we will evaluate whether the unmodified mRNA-

LPXs as such are capable of inducing DC-maturation. If they fail to do so, we will co-

incorporate several types of adjuvants (i.e. monophosphoryl lipid A (MPLA), CpG

oligodeoxynucleotides (ODN) and TriMix mRNA) and evaluate their effect on DC-maturation

in vitro.

Furthermore, we will evaluate whether the unmodified and immunomodulatory (i.e.

containing adjuvants) mRNA-LPXs are capable of stimulating DCs in vitro to trigger effective T

cell activation and proliferation. In order to evaluate this, we will perform an in vitro T cell

proliferation assay. Ultimately, we will perform a cytotoxic T lymphocyte (CTL) assay to

assess the capacity of particle loaded DCs to trigger antigen-specific CTL immune responses

in vitro.

16

Figure 2.1. Schematic representation of the co-delivery of both TAA encoding mRNA and

immunomodulating adjuvant to DCs via mRNA-adjuvant lipoplexes. This in order to achieve

TAA-loading and maturation of DCs and thereby enabling them to induce potent antigen-

specific T-cell immune responses against TAA expressing tumor cells.

17

3. MATERIALS AND METHODS

3.1. mRNA PREPARATION

3.1.1. Green fluorescent protein (GFP) mRNA

A bacterial culture of transformed E. coli with the plasmid (pGEM4Z/GFP/A64) was

grown in autoclaved LB-medium supplemented with antibiotics (ampicillin, 0.1 mg/ml;

Duchefa Biochem, Haarlem, The Netherlands). LB-medium was prepared by dissolving 10 g

NaCL (Lab M Limited, Bury, UK), 5 g Yeast extract (Lab M Limited) and 10 g Tryptone (Lab M

Limited) in 1 L distilled water. After 24 h, plasmids were isolated from the bacteria using the

QIAfilter plasmid purification kit according to the manufacturer’s instructions (Qiagen,

Venlo, The Netherlands). The obtained pDNA was either stored at -20°C in TE buffer (10 mM

Tris HCl, 1mM EDTA, pH 8.0; Fluka analytical, Buchs, Switzerland) or directly used for in vitro

mRNA transcription.

First, pDNA was linearized using the SpeI restriction enzyme (Promega, Leiden, The

Netherlands). Secondly, the linearized pDNA was used as a template for in vitro mRNA

transcription using the T7 mMessage mMachine kit (Ambion, Life Technologies, Ghent,

Belgium) containing T7 RNA polymerase, and the mRNA building blocks; the nucleotides and

a 7-methyl guanosine CAP analog structure. After 2 h mRNA transcription, a final purification

step was performed by DNase digestion of the remaining DNA, LiCl precipitation and the

obtained mRNA was washed with 70% ethanol. The mRNA concentration was determined

with the Nanodrop 2000 (Thermo Scientific, Zellik, Belgium), by measuring the absorbance at

260 nm and the mRNA was stored at -80°C in aliquots of 1µg/µl. A RNAse inhibitor (1U/µl)

was added to avoid rapid degradation (RNasin® Plus RNase inhibitor, Promega).

Figure 3.1. Structural elements of in vitro transcribed mRNA The in vitro transcribed mRNA

contains a 5’ cap, 5′and 3′ untranslated regions (UTRs), a coding region (i.e. open reading

frame (ORF)) and a poly (A) tail. (Sahin et al., 2014)

18

3.1.2. Ovalbumin (OVA) mRNA

Ovalbumin mRNA was kindly donated by the Laboratory of Molecular and Cellular

Therapy (LMCT; Vrije Universiteit Brussel, Brussels, Belgium).

3.2. LIPOPLEX PREPARATION

3.2.1. Unmodified mRNA-LPXs

3.2.1.1. Lipid stock solutions

The cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, MW 699.0

g/mol) was purchased from Avanti Polar Lipids (Alabaster, AL) and solved in chloroform

(CHCl3) at a concentration of 25 mg/ml. (figure 3.2.)

Figure 3.2. Molecular structure of DOTAP

The helper lipid DOPE (dioleoylphosphatidylethanolamine; MW 744.0 g/mol),

purchased from Avanti Polar Lipids was solved in CHCl3 to obtain a solution with a

concentration of 25 mg/ml. (figure 3.3.)

Figure 3.3. Molecular structure of DOPE

Cholesterol (MW 386,7 g/mol) was purchased from Sigma-Aldrich (Bornem, Belgium)

and solved in CHCl3 at a concentration of 20 mg/ml. (figure 3.4.)

Figure 3.4. Molecular structure of cholesterol

19

3.2.1.2. Liposome preparation

Cationic liposomes composed of DOTAP, combined with DOPE or cholesterol were

prepared by transferring the appropriate volumes of lipid stock solutions into a sterile

bottom round flask. (Table 3.1. and 3.2.) A lipid film was formed on the surface of the

bottom round flask by evaporation of the chloroform using nitrogen gas. Subsequently, the

lipid film was rehydrated in 500µl RNase-free water (Ambion) and vortexed to dissolve the

lipid film completely. Finally, the liposomes were sonicated in a bath sonicator (Branson

2510, Branson Ultrasonics, Dansbury, USA).

The commercial cationic liposome reagent, Lipofectamine® RNAiMAX (RNAiMAX), was

purchased from Invitrogen (Life technologies, Merelbeke, Belgium). The lipid concentration

of this commercial reagent was not provided by the manufacturer.

Table 3.1. DOTAP:DOPE liposomes: lipid mixture composition.

Lipid Concentration Used volume

DOTAP

DOPE

25 mg/ml

25 mg/ml

70 µl

74.4 µl

Table 3.2. DOTAP:CHOL liposomes: lipid mixture composition.

Lipid Concentration Used volume

DOTAP

Cholesterol

25 mg/ml

20 mg/ml

70 µl

72.5 µl

3.2.1.3. mRNA loading of the liposomes

The mRNA lipoplexes were generated by mixing 1µg negatively charged OVA or GFP

mRNA with the appropriate volumes of cationic liposomes in different cationic lipid-to-

mRNA charge (N/P) ratios (DOTAP:DOPE and DOTAP:CHOL mRNA-LPXs) or µl RNAiMAX/µg

mRNA ratios (RNAiMAX mRNA-LPXs) in HEPES (20 mM, PH 7.4; Sigma-Aldrich). Subsequently,

the lipoplexes were incubated for 15 min at room temperature.

20

The N/P ratio of a lipoplex is defined as the molar ratio of positively charged amine

groups of the cationic liposomes to the negatively charged phosphate groups of mRNA. Due

to the fact that the lipid concentration of the commercial reagent Lipofectamine® RNAiMAX

was not provided by the manufacturer, we could not determine the appropriate volume

needed to deliver a certain quantity of positively charged amine groups needed to obtain a

certain N/P ratio. Therefore, we quantified the composition of the lipoplexes based on the

amount of commercial Lipofectamine® RNAiMAX reagent (µl) used per µg mRNA (i.e. µl

RNAiMAX/µg mRNA ratio).

3.2.2. Immunomodulatory DOTAP:CHOL mRNA-LPXs

Lipoplexes containing the immunomodulator monophosphoryl lipid A (MPLA) were

prepared by embedding the appropriate amount of MPLA into the liposomal membrane.

More specifically, during liposome preparation, 0.5 mole % MPLA (Invivogen, Toulouse,

France) was added to the DOTAP and cholesterol lipid mixture. After liposome generation,

the corresponding lipoplexes were prepared as described above.

To prepare DOTAP:CHOL LPXs which include both OVA or GFP mRNA and TriMix

mRNA (i.e. 3 mRNA sequences encoding CD40L, caTLR4, CD70; eTheRNA, Kortenberg,

Belgium), DOTAP:CHOL liposomes were loaded with equal amounts (i.e. 0.5 µg) of the 4

different mRNA sequences according to 3.2.1.3.. To maintain the optimal N/P ratio, using a

total amount of 2µg mRNA instead of 1, the amount of liposomes was doubled.

A third type of immunomodulatory DOTAP:CHOL mRNA-LPXs was generated by

including equal amounts (i.e. 1 µg) of OVA or GFP mRNA and CpG

oligodeoxynucleotides (CpG ODN; Invivogen, Toulouse, France) into the DOTAP:CHOL

liposomes according to 3.2.1.3.. Once again the amount of liposomes was doubled to

maintain the optimal N/P ratio and complete complexation.

3.3. LIPOPLEX CHARACTERIZATION

The obtained lipoplexes were characterized by determination of the hydrodynamic

diameter as well as the zeta potential using the Zetasizer NanoZS (Malvern Instruments Ltd,

Worcestershire, UK). Therefore, the lipoplex solution was added to 1 ml of HEPES buffer

which was then loaded into a disposable folded capillary cell and transferred into the

Zetasizer NanoZs.

21

3.3.1. Dynamic light scattering (DLS)

The hydrodynamic diameter was determined by applying the Dynamic Light

Scattering (DLS) technique. This technique is based on the phenomenon of Brownian

motion, which is defined as the random motion of particles due to bombardment by

surrounding solvent molecules. When particles are illuminated by a laser beam, Brownian

motion of the suspended particles causes intensity fluctuations of the scattered light. By

analyzing these fluctuations, the velocity of the Brownian motion, otherwise defined as the

translational diffusion coefficient, can be determined. Knowing this velocity, the

hydrodynamic diameter can be calculated using the Stokes-Einstein relation.

3.3.2. Zeta potential measurements

The determination of the lipoplex surface charge was evaluated by means of zeta

potential measurements. The basis of this method is the formation of an electrical double

layer around each particle. When a particle in solution has a net charge, a strongly bound

layer of oppositely charged solvent ions arises around the particle, referred to as the Stern

layer. More distant to the particle, ions are more loosely associated creating a diffuse outer

layer. When a particle moves, a boundary exists between the ions in the diffuse layer that

move with the particle and the ions that stay with the bulk dispersant. The electrostatic

potential at this boundary is called the zeta potential.

For the measurement of the zeta potential using the Zetasizer NanoZS, an electric

field is applied across the sample causing charged particles to move toward the electrode

with the opposite charge. The velocity of the particle motion, otherwise defined as the

electrophoretic mobility, is measured by light scattering techniques (i.e. a patented laser

interferometric technique called M3-PALS (Phase analysis Light Scattering)). The zeta

potential can now be calculated using the Henry equation.

3.4. mRNA COMPLEXATION

To evaluate the mRNA incorporation into the different lipoplexes at the different µl

RNAiMAX/ µg mRNA or N/P ratios, agarose gel electrophoresis was performed. The 1%

agarose gel was prepared using 1 g agarose (Gibco-Invitrogen) dissolved in 100 ml TBE

buffer (10,78 g TRIS (Sigma-Aldrich, St. Louis, USA), 5,58 g Boric acid (Fluka analytical,

Buchs, Switzerland), 0,74g EDTA (Merck, Darmstadt, Germany)). After heating to boiling

22

point, 10 µl Gel Red Nucleic Acid Gel stain (10,000 x in water; Biotium, Hayward, California)

was added and the gel was poured into a comb containing gel holder where the gel was

allowed to set for 30 min. 5 µl 5x loading buffer (Ambion, Life Technologies, Ghent,

Belgium) was added to the lipoplexes and the samples were loaded into the wells.

Electrophoresis was performed for 20 min at 100 V. To visualize the gel, it was placed onto

a Bio-Rad UV Transilluminator 2000 system (Bio-rad, Temse, Belgium) and a photograph

was taken using a Kodak DS electrophoresis documentation and analysis system and the

Kodak digital science 1 ID LE 3.0 software.

3.5. CELL CULTURE

Primary murine bone marrow-derived DC (BM-DC) cultures were obtained from

C57BL/6 mice (female; Harlan laboratories, Gannat, France) housed under SPF conditions

according to the Belgium law and the local Ethical Committee. Mice were euthanized and

the bone marrow was flushed from the tibia and femur of the hind limbs with DPBS (Gibco-

Invitrogen, Merelbeke, Belgium). Bone marrow cells were collected and frozen in

cryomedium (FetalCloneTM I serum ( FCI; HycloneTM, Pierce, Rockford, IL, USA), 2% glucose

(Sigma-Aldrich) and 10% DMSO (Sigma-Aldrich); one mice leg per freezing) to -80°C. For BM-

DC generation, a bone marrow freezing was thawed and obtained cells were cultured in

culture dishes (100 mm Not TC-Treated polystyrene Culture Dishes; Corning, Amsterdam,

The Netherlands) in cell culture medium. The used medium was RPMI 1640 (Gibco-

Invitrogen) with 1% penicillin/streptomycin/L-glutamine (Gibco-Invitrogen), 50µM β-

mercapthoethanol (Gibco-Invitrogen) and 5 % FetalCloneTM I serum (FCI; HycloneTM).

Differentiation of monocytes into BM-DCs was attained by adding GM-CSF (20 ng/ml;

Peprotech, Rock Hill, NJ, USA). After an incubation (37°C, 5% CO2) of 3 days, 15 ml culture

medium supplemented with GM-CSF (40 ng/ml; Peprotech) was added to the culture. On

day 5, the cells were collected from the culture dishes and counted using the Bürker

counting chamber after trypan blue staining to exclude death cells. Then, the collected cells

were centrifuged (6 min, 400 rcf) after which they were resuspended in GM-CSF

supplemented (20 ng/ml) culture medium to obtain a 1 x 106 cell/ml cell suspension. Finally,

the cells were seeded and incubated in 24 well plates at 5 x 105 cells per well.

23

3.6. LIPOPLEX LOADING OF DCs

At day 6, the seeded BM-DCs in the 24-well plates were loaded with lipoplexes and

incubated at 37°C. To evaluate the influence of serum, loading was performed in two

different media, namely culture medium (5% FCI serum) and Opti-MEM® (i.e. a serum

reduced medium).

3.6.1. Loading in culture medium (5% FCI serum)

Seeded BM-DCs were loaded by adding the prepared lipoplexes into the wells which

already contained BM-DCs in culture medium (5% FCI serum). More specifically, for the

experiments with unmodified LPXs, 1µg OVA or GFP mRNA incorporated into the lipoplexes

was added per well. For experiments using immunomodulatory LPXs, the quantities

described in 3.2.2. were added per well.

3.6.2. Loading in serum reduced medium (Opti-MEM®)

To collect the non-adherent cells, cell culture medium was collected from the wells

and centrifuged for 5 min at 400 rcf. In the meantime the prepared lipoplexes were diluted

with Opti-MEM® and the obtained lipoplex solution was added into the wells (quantities

were analogues to 3.6.1.). Finally, the collected non-adherent cells were re-added to the

wells. After 2 h of incubation, supernatant was removed and replaced by cell culture

medium.

3.7. TRANSFECTION EFFICIENCY AND MATURATION STATUS

At day 7, the transfection efficiency and maturating capacity of the different

lipoplexes was quantified by flow cytometry, using a FACSCaliburTM (BD Pharmingen,

Erembodegem, Belgium). Flow cytometry is a technology used to characterize different

properties (e.g. size, granularity and fluorescence) of single cells as they flow in a fluid

stream through a laser beam. When a sample is introduced into the flow cytometer, the

injected sample fluid is hydrodynamically focused into a single-file stream by the fluidics

system of the flow cytometer. As a result, the cells pass one-by-one through a laser beam

(i.e. blue or red diode laser). When the laser beam strikes the cell, laser light is scattered in

different directions. The scattered light is collected on a photodetector and converted into

an electronic signal. The magnitude of forward scattered light (FSC) is proportional to the cell

size, while side scattered light (SSC) is proportional to cell granularity. Furthermore, in order

24

to evaluate other cell characteristics, cells can be labeled with fluorophores. When laser light

of the correct wavelength strikes a fluorescently labeled cell, a fluorescent signal is emitted

which can be detected by one of the four detectors. In this way, the fluorescence of each cell

can be evaluated.

Figure 3.5. Flow cytometer: instrument overview (bcrf.ucsd.edu)

Previous to analysis by flow cytometry, the cells were collected from the wells and

transferred to a FACS tube. Cells were washed twice using FACS buffer (DPBS (Gibco-

Invitrogen), 1% BSA (Sigma-Aldrich), 0.09% Sodium azide (Sigma-Aldrich)) and the population

of DCs in the cell culture was stained using Allophycocyanin (APC)-labeled anti-mouse CD11c

antibodies (1.5µl/sample, eBioscience, San Diego, USA). Transfection efficiency

measurements were based on GFP expression. To examine the DC maturation status, anti-

mouse CD40-Phycoerythrin (PE) antibodies (1.5µl/sample, eBioscience) and anti-mouse

CD86-PE antibodies (1.5µl/sample, eBioscience) were used to stain DCs with upregulated

CD40 and CD86 maturation markers respectively. After incubation at 4°C for 45 min, the

samples were washed twice with FACS buffer. Finally, after resuspension in 250µl FACS, the

samples were analyzed. The obtained data were processed using the BD CellQuest ProTM or

FlowJo software.

3.8. IN VITRO T CELL PROLIFERATION ASSAY

In order to evaluate the capacity of particle loaded DCs to prime antigen-specific

CD8+ T cells, an in vitro T cell proliferation assay was performed. First, OT-I cells, which

express a transgenic T cell receptor that recognizes the MHC-I restricted ovalbumin (OVA)

25

peptide SIINFEKL, were isolated from OT-I spleen freezings (LMCT, VUB) using the EasySep™

Mouse CD8+ T Cell Isolation Kit with an EasySep™ magnet (Stemcell Technologies,

Vancouver, Canada). The isolated CD8+ OT-I cells, resuspended in isolation culture medium,

were then counted using the Bürker counting chamber after trypan blue staining. The cells

were then centrifuged (5 min, 500 rcf) and washed using PBS/0.1% BSA (Amresco, Solon,

USA). Subsequently, the cells were fluorescently labeled with carboxyfluorescein

succinimidyl ester (CFSE) (1ml of 5µM solution of CFSE in PBS/0.1% BSA per 1x106 cells;

eBioscience) and incubated at 37°C for 20min. After a second wash and counting step, the

stained OT-I cells were seeded in a U-bottom 96 well plate at 1x105 T cells/well and co-

cultured with collected DCs seeded at 1x104 DCs/well. Previous to seeding, the DCs were

cultured and transfected according to 3.5. and 3.6.2. with either unmodified or

immunostimulatory OVA-loaded particles. Untreated and SIINFEKL pulsed DCs were used as

a negative and positive control respectively. After incubation overnight, the appropriate

samples were matured with LPS for 4h after which the samples were ready for seeding.

After 5 days of co-culturing, the cells were collected and transferred to a FACS tube.

The samples were washed twice using FACS buffer after which they were stained with APC-

labeled anti-mouse CD8 antibodies (BD Pharmingen) and incubated at 4°C for 30 min. After

incubation, the samples were washed twice. Finally, after resuspension in 250µl FACS, the

samples were analyzed. The obtained data were processed using the BD CellQuest ProTM or

FlowJo software.

3.9. IN VITRO CYTOTOXIC T LYMPHOCYTE (CTL) ASSAY

In order to assess the capacity of particle loaded DCs to trigger antigen-specific CTL

immune responses, an in vitro CTL assay was performed. CD8+ OT-I cell isolation and

staining, DC transfection and subsequent co-culturing were executed according to 3.8.. After

5 days of co-culturing, the samples were challenged with both EL4 tumor cells (i.e. control

cells) and E.G7-OVA tumor cells (i.e. target cells) in a ratio of 7 to 1 (T cells to tumor cells).

The added EL4 tumor cells were labeled with cell proliferation dye eFluor 670 (CPD;

eBioscience) at low intensity (0.03 µM). In contrast, the E.G7-OVA tumor cells were labeled

with CPD at high intensity (0.6 µM). In order to accomplish this, the EL4 and E.G7-OVA tumor

cells were first counted using the Bürker counting chamber after which the appropriate

amount of EL4 and E.G7-OVA tumor cell suspension was collected. The obtained cell pellets

26

were resuspended in 10 ml PBS/0.1%BSA and the cells were then fluorescently labeled with

either 0.03µM CPD or 0.6µM CPD for EL4 and E.G7-OVA cells respectively. After incubation

(37°C, 20 min), the cells were washed with PBS/0.1%BSA and finally the appropriate amount

(i.e. to obtain a 7 to 1 ratio per well) of stained EL4 and E.G7-OVA cells, resuspended in 100µl

cell culture medium, was added to each well of the co-culture. After 4h of co-incubation, the

cells were collected and transferred to a FACS tube. The samples were washed once using

FACS buffer and finally, after resuspension in 250µl FACS, the samples were analyzed. The

obtained data were processed using the BD CellQuest ProTM or FlowJo software.

The percentage specific lysis of target cells was calculated as:

(1 − ((%CPDhigh / %CPDlow)treated / (%CPDhigh / %CPDlow)untreated) × 100%.

3.10. CYTOKINE SECRETION

The levels of secreted IL12-p70 and IL-10 were measured via enzyme-linked

immunosorbent assay (ELISA; all Ready-SET-Go!® ELISA kits, eBioscience) following the

manufacturer’s instructions. First a plastic 96 well plate was coated with anti-cytokine

capture antibodies. Subsequently, the samples (100µl of 2-fold diluted supernatant) were

added into the coated wells. After 2h incubation at room temperature, biotin-conjugated

anti-cytokine detection antibodies were added followed by avidin-horseradish

peroxidase (HRP)-conjugates. Finally, the chromogenic substrate 3,3′,5,5′-

tetramethylbenzidine (TMB) was added to each well (10 min). After the addition of stop-

solution (1M H3PO4), the optical density (OD) was measured using the EnVision Multilabel

Reader at 450nm.

3.11. STATISTICAL ANALYSIS

All date are presented as mean ± standard deviation. Two samples were compared

using a two-sided unpaired Student’s t-test. Statistical significance was assessed at P < 0,05.

Presented data are representative for at least 2 independent experiments performed on

BM-DCs derived from different donor mice, except for the T cell proliferation- and CTL assay.

27

4. RESULTS

4.1. PARTICLE CHARACTERIZATION

In a first step, the mRNA inclusion into the DOTAP:DOPE, DOTAP:CHOL and RNAiMAX

mRNA-LPXs was evaluated as a function of different N/P (DOTAP:DOPE and DOTAP:CHOL) or

µl RNAiMAX/µg mRNA (RNAiMAX) ratios by agarose gel electrophoresis. Since free mRNA

will not reach the DCs due to rapid mRNA degradation and inefficient cell uptake, complete

incorporation of the mRNA is desirable. For DOTAP:DOPE, N/P ratios ≥ 2.5 allowed complete

complexation of the mRNA. Nearly similar results were seen with DOTAP:CHOL mRNA-LPXs

showing complete incorporation of the mRNA at N/P ratios ≥ 2. For RNAiMAX, the results

demonstrate that a ratio ≥ 5 is needed to achieve complete mRNA inclusion into the mRNA-

LPXs. (Figure 4.1.)

Figure 4.1. Agarose gel electrophoresis of (A.) DOTAP:DOPE, (B.) DOTAP:CHOL and (C.)

RNAiMAX mRNA-LPXs with differing N/P or µl RNAiMAX/ µg mRNA ratios in HEPES.

28

Based on these results, we decided to continue further experiments with DOTAP:DOPE and

DOTAP:CHOL mRNA-LPXs at a N/P ratio of 2.5. For RNAiMAX, two ratios (2 and 5) were

included for further experiments. In fact, although incomplete mRNA-incorporation was

observed at ratio 2, preliminary data showed a higher transfection efficiency in Opti-MEM®

upon the use of RNAiMAX mRNA-LPXs ratio 2 as compared to RNAiMAX mRNA-LPXs at ratio

5, possibly due to a diminished toxicity at this ratio.

In a second step, we wanted to assess whether complete mRNA incorporation

remained in the presence of serum. Indeed, it is well known that due to interactions

between the lipoplexes and negatively charged serum components, the content of the LPXs

can be released. In order to evaluate this, a second agarose gel electrophoresis was

performed. This time, FetalCloneTM I serum (FCI) was added to the generated mRNA-LPXs

obtaining a FCI concentration of 50%. After an incubation time of 30′ mRNA incorporation

was evaluated. Figure 4.2. shows that complete mRNA complexation persists for

DOTAP:DOPE and DOTAP:CHOL mRNA-LPXs at N/P 2.5. In contrast, free mRNA was detected

with the RNAiMAX mRNA-LPXs at ratio 5 indicating incomplete mRNA incorporation in the

presence of serum.

Figure 4.2. Agarose gel electrophoresis of DOTAP:DOPE (N/P 2.5), DOTAP:CHOL (N/P 2.5)

and RNAiMAX (µl RNAiMAX/ µg mRNA 2 and 5) mRNA-LPXs in the presence of 50% FCI.

C.

29

In a second step, the different lipoplexes were characterized by the determination of

the hydrodynamic diameter as well as the zeta potential using the Zetasizer NanoZS.

Characterizing size and zeta potential is desirable considering the fact that these two

parameters are capable of affecting the transfection efficiency of the generated mRNA-LPXs.

More specifically, particle size can determine the pathway of entry of the LPXs, while the

zeta potential has an impact on the interaction with the cell membrane.(Rejman et al., 2004)

In fact, the latter is facilitated when particles possess a positive surface charge.

The particle size distributions of the different LPXs are illustrated in Figure 4.3.A..

DOTAP:DOPE and DOTAP:CHOL mRNA-LPXs had a mean particle size of 144.1 ± 0.6 nm and

154.0 ± 0.8 nm with a polydispersity index (PDI) of 0.13 ± 0.02 and 0.13 ± 0.02 respectively.

Notably, for RNAiMAX mRNA-LPXs at ratio 5, large aggregates were observed with a mean

particle size of 3136.0 ± 144.2 nm and a PDI of 0.68 ± 0.21. In contrast, for RNAiMAX mRNA-

LPXs at ratio 2, the observed mean particle size of 134.3 ± 4.5 nm with a PDI of 0.15 ± 0.02

more closely resembled the mean particle size observed for DOTAP:DOPE and DOTAP:CHOL

mRNA-LPXs.

As expected, a positive average zeta potential of 42.9 ± 1.3 mV and 47.2 ± 1.8 mV was

observed with DOTAP:DOPE and DOTAP:CHOL mRNA-LPXs. In contrast, RNAiMAX mRNA-

LPXs at ratio 2 and 5 had a negative zeta potential of -47.4 ± 3.0 mV and -27.75 ± 2.0 mV

respectively. (Figure 4.3.B.)

Figure 4.3. Graphical representation of (A.) the average size and (B.) the average zeta

potential of the different mRNA-LPXs in HEPES (n=3).

30

4.2. TRANSFECTION EFFICIENCY

In a next step, we assessed the capacity of the unmodified DOTAP:DOPE, DOTAP:CHOL

and RNAiMAX mRNA-LPXs to efficiently introduce mRNA encoded proteins into murine bone

marrow derived DCs in vitro. To evaluate this, we transfected seeded BM-DCs with

DOTAP:DOPE, DOTAP:CHOL and RNAiMAX-LPXs containing mRNA encoding the reporter

protein GFP. To determine the influence of serum on the transfection efficiency (TE),

transfection was performed in two different media, namely Opti-MEM® (a serum reduced

medium) and culture medium (5% FCI serum).

Figure 4.4. Percentage GFP expressing-DCs 24h after loading with DOTAP:DOPE (N/P 2.5),

DOTAP:CHOL (N/P 2.5) and RNAiMAX (µl RNAiMAX/ µg mRNA 2 and 5) mRNA-LPXs

(n=3).*p<0.05, **p<0.01, ***p<0.001.

The results in Figure 4.4. illustrate that DOTAP:DOPE mRNA-LPXs (N/P 2.5) are efficient

particulate systems to deliver GFP mRNA to BM-DCs in serum reduced medium. However, in

the presence of serum (5% FCI), a total loss in TE was observed. In contrast, the TE of

DOTAP:CHOL mRNA-LPXs (N/P 2.5) did not significantly alter in the presence of serum (5%

FCI). Notably, for RNAiMAX mRNA-LPXs, a higher TE in serum-reduced medium could be

observed for mRNA-LPXs at ratio 2 as compared to mRNA-LPXs at ratio 5. A high toxicity

observed for the mRNA-LPXs at ratio 5 could possible explain the low TE in Opti-MEM®

observed at this ratio. In serum, the TE obtained with RNAiMAX mRNA-LPXs exceeds the TE

observed for DOTAP:DOPE mRNA-LPXs. However, it does not surpass the TE of DOTAP:CHOL

mRNA-LPXs. These results demonstrate that DOTAP:CHOL mRNA-LPXs are the most optimal

31

delivery systems in the presence of serum. Based on these observations, we decided to

continue further experiments with DOTAP:CHOL mRNA-LPXs at a N/P ratio of 2.5.

4.3. DENDRITIC CELL ACTIVATION

4.3.1. DC activation by unmodified DOTAP:CHOL mRNA-LPXs

Besides efficient delivery of mRNA encoded proteins, the lipid-based mRNA delivery

system has to be capable of stimulating full maturation of DCs in order to induce potent

cytotoxic T-cell responses. During DC-maturation, a variety of changes are initiated, including

an increased expression of maturation markers (e.g. CD40, CD80, CD86) and the production

of cytokines (e.g. IL-12p70). Thus, to estimate the effect of mRNA-LPXs on the maturation

status, the expression of different maturation markers as well as the secretion of several

types of cytokines could be evaluated.

To assess the influence of unmodified DOTAP:CHOL mRNA-LPX transfection on the

DC maturation status, the expression the maturation marker CD40 was evaluated by means

of flow cytometry. As a negative control, we used untreated (blank) dendritic cells. As a

positive control, untreated DCs were pulsed with bacteria-derived lipopolysaccharide (LPS)

(i.e. TLR4 agonist), which is a well-known maturation-stimulus. Furthermore, to evaluate

whether transfected DCs are still capable of responding to maturation-stimuli, another

control group was included. Within this group, DCs were transfected with unmodified

DOTAP:CHOL mRNA-LPXs after which LPS was added to the culture medium.

The results in Figure 4.5. demonstrate that unmodified DOTAP:CHOL mRNA-LPXs as

such, induce only a very small, but significant shift in CD40 expression. As compared to the

positive control, this shift in expression is only limited. Furthermore, the data show a

significantly increased CD40 expression upon addition of LPS to particle-loaded DCs. This

indicates that the DCs preserve the capacity to respond to maturation-stimuli, in a similar

way to blank cells. These observations prompted us to include additional adjuvants into the

mRNA-LPXs to further increase the DC maturation-status.

32

Figure 4.5. Influence of unmodified DOTAP:CHOL mRNA-LPXs (N/P 2.5) on the CD40

maturation marker expression. The percentage CD40 expressing-DCs, 24h after loading

with the different samples (N/P 2.5), is represented in (A.) (n=3). *p<0.05, ***p<0.001.

Representative histograms for these samples are shown in (B.).

4.3.2. DC activation by immunomodulatory DOTAP:CHOL mRNA-LPXs

With regard to the development of mRNA-LPXs which stimulate full DC-maturation,

three types of adjuvants were included into the DOTAP:CHOL mRNA-LPXs, namely

monophosphoryl lipid A (MPLA), TriMix mRNA or CpG oligodeoxynucleotides (CpG ODN).

Monophosphoryl lipid A (MPLA), a less-toxic derivative of bacteria-derived LPS (i.e. TLR4

agonist) was embedded into the liposomal membrane at a mole percentage of 0.5. In a

second type of immunomodulatory DOTAP:CHOL mRNA-LPXs, we included TriMix mRNA.

TriMix mRNA encodes CD40 ligand (CD40L), a constitutive active TLR4 and CD70. Via

exploratory research, we determined that optimal transfection and maturation can be

obtained by loading the DOTAP:CHOL liposomes with equal amounts (i.e. 0.5 µg) of the 4

different mRNA sequences. (data not shown) To maintain the optimal N/P ratio of 2.5, using

a total amount of 2 µg mRNA instead of 1 µg, the amount of liposomes was doubled. In a last

type of immunomodulatory DOTAP:CHOL mRNA-LPXs, we co-incorporated CpG ODN. CpG

ODN are synthetic oligodeoxynucleotides (ODN) that contain CpG motifs. Mimicking

bacterial DNA, these CpG motifs are recognized by TLR9. CpG containing LPXs were

generated by including equal amounts (i.e. 1 µg) of GFP mRNA and CpG

33

oligodeoxynucleotides (CpG ODN) into the DOTAP:CHOL liposomes. Once again the amount

of liposomes was doubled to maintain the optimal N/P ratio of 2.5. Complete complexation

of mRNA and adjuvant (TriMix mRNA or CpG ODN) was confirmed by agarose gel

electrophoresis. (Data not shown)

To assess whether the different adjuvants, co-incorporated into the DOTAP:CHOL

mRNA-LPXs, are capable of further increasing the DC maturation status, we evaluated the

expression of both the DC-maturation marker CD40 and CD86 24h post transfection by

means of flow cytometry. The same positive and negative control group was used as

described in 4.3.1.. As demonstrated in Figure 4.6., CpG ODN and MPLA containing

immunomodulatory LPXs induced a major significant shift in CD40 expression as compared

to the unmodified DOTAP:CHOL mRNA-LPXs. In contrast, addition of TriMix mRNA induced

only a minor additional increase in the expression of CD40.

Figure 4.6. Influence of immunomodulatory DOTAP:CHOL mRNA-LPXs (N/P 2.5) on the

CD40 maturation marker expression. The percentage CD40 expressing-DCs, 24h after

transfection with the different samples (N/P 2.5), is represented in (A.) (n=3). ** p<0.01,

***p<0.001. Representative histograms for these samples are represented in (B.).

34

With regard to the expression of the maturation marker CD86, similar observations

could be made. DOTAP:CHOL mRNA-LPXs including CpG ODN or MPLA had a major

significant effect on the CD86 expression as compared to the unmodified DOTAP:CHOL

mRNA-LPXs. In contrast, addition of TriMix mRNA induced only a minor but significant

upregulation of CD86. Unlike the data we obtained on CD40 expression, we did not observe

a significant shift in CD86 expression when DCs were loaded with unmodified DOTAP:CHOL.

(Figure 4.7.)

Figure 4.7. Influence of immunomodulatory DOTAP:CHOL mRNA-LPXs (N/P 2.5) on the

CD86 maturation marker expression. The percentage CD86 expressing-DCs, 24h after

transfection with the different samples (N/P 2.5), is represented in (A.) (n=3). ** p<0.01,

***p<0.001. Representative histograms for these samples are represented in (B.)

Besides an increased expression of co-stimulatory molecules, a second aspect that

can be observed during DC maturation is the production and secretion of cytokines. We

quantified the secretion of the two different cytokines IL-12p70 and IL-10 via ELISA. IL-12p70

is a pro-inflammatory cytokine which is crucial for T cell activation. On the other hand, IL-10

is an anti-inflammatory cytokine that plays an important role in immune tolerance.

Therefore, the ratio of IL-12p70 and IL-10 is often used as a measure for the stimulatory

capacity of DCs.

35

Figure 4.8. Graphical representations of the levels of IL-12p70 (A.) and IL-10 (B.) secreted

by DCs after loading with either unmodified DOTAP:CHOL mRNA-LPXs or different types of

immunomodulatory DOTAP:CHOL mRNA-LPXs (n=3). The ratio IL-12p70/IL-10 is

represented in (C.) (n=9).*<0.05,** p<0.01, ***p<0.001.

The data in Figure 4.8.A. illustrate a significant increase in IL-12p70 secretion 24h

after DC-loading with DOTAP:CHOL mRNA-LPXs including either CpG ODN or MPLA.

Moreover, the secretion of IL-12p70 was significantly higher when DCs were loaded with

CpG-modified mRNA-LPXs in comparison with LPS pulsed DOTAP:CHOL loaded-DCs. In

contrast, when MPLA was included into the mRNA-LPXs, no additional increase in IL-12p70

concentration was detected as compared to DOTAP:CHOL loaded-DCs matured with LPS. No

increase in IL-12p70 secretion (i.e. ≥ the detection limit of 15pg/ml) was detected with

unmodified DOTAP:CHOL and TriMix-modified mRNA-LPXs. With regard to the secretion of

IL-10, we also detected a significant increase when DCs were loaded with DOTAP:CHOL

mRNA-LPXs containing CpG or MPLA. However, for both immunomodulatory mRNA-LPXs,

the concentrations of IL-10 were still significantly lower than when LPS was added to induce

maturation of DOTAP:CHOL loaded-DCs. Furthermore, no increase in IL-10 concentration

36

was detected with unmodified DOTAP:CHOL and TriMix-modified mRNA-LPXS. (Figure 4.8.B.)

In conclusion, with regard to the ratio of IL-12p70 and IL-10, the most optimal ratio was

observed for CpG containing DOTAP:CHOL mRNA-LPXs.

4.3.3. Influence on transfection

To evaluate whether the co-incorporation of the different adjuvants into the

DOTAP:CHOL mRNA-LPXs could influence the TE, we compared the TE of unmodified

DOTAP:CHOL mRNA-LPXs with the TE of the different immunomodulatory DOTAP:CHOL

mRNA-LPXs by flow cytometry. In Figure 4.9., the results of this experiment are illustrated.

The data show that both unmodified and immunomodulatory mRNA-LPXs were capable of

inducing expression of the reporter protein GFP. Moreover, co-incorporation of CpG or

MPLA into the DOTAP:CHOL mRNA-LPXs did not result in a diminished transfection

efficiency. In contrast, a significant decrease in transfection efficiency could be observed

when TriMix mRNA was co-incorporated into the LPXs.

Figure 4.9. Graphical representation of the transfection efficiencies of both unmodified

DOTAP:CHOL mRNA-LPXs and immunomodulatory DOTAP:CHOL mRNA-LPXs (n=3).

*p<0.05, **p<0.01.

37

4.4. INDUCTION OF CD8+ T CELL PROLIFERATION

In the previous chapters, we have shown that it is possible to develop a particle that

is capable of (a) introducing mRNA encoded proteins into murine BM-DCs as well as (b)

increasing the DC-maturation status. To verify whether particle loaded-DCs truly possess the

ability of inducing antigen-specific CD8+ T cell activation and proliferation, an in vitro OT-I

cell proliferation assay was performed. In this assay, OT-I cells carrying a T cell receptor that

recognizes the MHC-I restricted ovalbumin (OVA) peptide SIINFEKL, are labeled (CFSE) and

co-cultured with DCs which are transfected with different types of OVA mRNA-LPXs. To

evaluate antigen-specificity, a negative control group was co-cultured with DCs transfected

with GFP mRNA-LPXs. As a positive control, a group of DCs was co-cultured with mature

SIINFEKL-DCs. After 5 days, the percentage proliferating OT-I cells was evaluated by

measuring the fluorescence intensity using the FACSCaliburTM. In fact, with each cell division

of an original mother cell, the fluorescence of this mother cell will be distributed amongst

the daughter cells. As a result, the decrease in fluorescence intensity can be linked with the

proliferation of OT-I cells.

Figure 4.10. In vitro CD8+ T cell proliferation triggered by DCs transfected with unmodified

DOTAP:DOPE or DOTAP:CHOL mRNA-LPXs or different types of immunomodulatory

DOTAP:CHOL mRNA-LPXs (n=2). (A.) Representative histograms for these samples are

provided in (B.)

38

Extensive T cell proliferation was induced by both unmodified DOTAP:CHOL mRNA-

LPX- and immunodulatory mRNA-LPX-transfected DCs, as shown in Figure 4.10. Interestingly,

the capacity to activate CD8+ T cells differed greatly between the DOTAP:CHOL and

DOTAP:DOPE loaded DCs which pinpoints the importance of a serum resistant carrier for the

delivery of the mRNA. We could not yet detect a significant difference in CD8+ T cell

activating capacity between either DCs transfected with unmodified DOTAP:CHOL LPXs or

these transfected with the CpG, MPLA or TriMix containing mRNA-LPXs. However, when

looking at the representative histograms, a trend towards more extensive T cell proliferation

can be observed upon co-incorporation of the above mentioned adjuvants. Nevertheless, we

must point out the necessity to repeat this experiment since we were only able to perform

this experiment once with a low quantity of samples. By repeating the experiment, possible

significant differences could yet become clear. Lastly, with regard to antigen-specificity, we

must mention that a slight increase in T cell proliferation was observed when DCs were

loaded with unmodified or immunomodulatory GFP-mRNA-LPXs.

4.5. INDUCTION OF ANTIGEN SPECIFIC CYTOTOXIC T CELL (CTL) RESPONSES

Besides inducing antigen-specific CD8+ T cell activation and proliferation, particle

loaded DCs should be able to trigger antigen-specific lysis of tumor cells by the induction of

antigen-specific CTL immune responses. To verify this, an in vitro CTL assay was performed

using OVA as our model antigen. In this assay, OVA-expressing tumor cells (i.e. E.G7-OVA,

target cells) and EL4 tumor cells (i.e. non-target cells) were labeled (CPD) at different

intensities and subsequently added to the co-cultures of particle loaded DCs and OT-I cells.

After 4h of co-incubation, the ratio of the E.G7-OVA target cells versus the EL4 control cells

was examined as a measure for antigen-specific lysis of tumor cells. The same control groups

as described in 4.4. were used.

The results in Figure 4.11. illustrate that only the use of a three component system

can induce antigen-specific lysis of E.G7-OVA cells. However, as compared to the positive

control group, the antigen-specific lysis induced by the different immunomodulatory mRNA-

LPXs was only limited. Moreover, we could not yet prove statistical significant differences

between the different immunomodulatory mRNA-LPXs. However, once again, we must point

out the necessity to repeat this experiment since we were only able to perform this

experiment once with a low quantity of samples. By repeating the experiment, possible

39

differences could yet become clear. Notably, although the co-incorporation of TriMix mRNA

into the mRNA-LPXs did result in a decrease in transfection efficiency and an only limited

upregulation of maturation markers, the observed T cell proliferation and CTL responses

observed with the corresponding LPXs were similar to those obtained with the other types of

immunomodulatory mRNA-LPXs.

Figure 4.11. Antigen-specific lysis of target cells via CTL responses triggered by DCs

transfected with unmodified DOTAP:DOPE or DOTAP:CHOL mRNA-LPXs or different types

of immunomodulatory DOTAP:CHOL mRNA-LPXs (n=2).

40

5. DISCUSSION

In this study, we aimed to design a lipid-based delivery tool that stimulates DCs in

vitro to induce potent cellular immune responses against specific TAAs. The development of

such a tool will be a major step forward towards developing in vivo applicable DC-vaccines

that might replace the expensive, patient-specific and labor-intensive ex vivo DC-vaccines.

In order to induce potent TAA-specific cellular immune responses, the lipid-based

delivery system has to (a) efficiently introduce antigenic information into DCs and (b)

stimulate full DC-maturation. A widely used method to deliver antigenic information to DCs

is the use of synthetic peptides that represent defined TAA-epitopes. However, this

approach is limited by a number of drawbacks including (a) a lack of characterized tumor

epitopes, (b) MHC restriction, (c) a short half-life of the peptide-MHC complexes and (d)

narrow immune responses. Several studies indicate that these limitations can be

circumvented when DCs are loaded with TAA-encoding mRNA. (Benteyn et al., 2014; Zhang

et al., 2002)

In a first phase of this study, we aimed to develop and characterize a delivery system

that is capable of efficiently introducing mRNA encoded proteins (e.g. TAAs) into BM-DCs in

vitro, especially in the presence of serum (i.e. optimal for in vivo applications). To date, in

vitro experiments assessing the transfection efficiency (TE) of nanoparticles are often

exclusively carried out in serum-free medium, making it very difficult to predict in which way

the nanoparticles will behave in vivo. In fact, serum has been reported to greatly decrease

the transfection efficiency of different types of LPXs in different cell types (e.g.

lipofectamine, DC-chol/DOPE, DOTMA). (Dodds et al., 1998; Li et al., 1999; Zhang and

Anchordoquy, 2004) Consistent with these studies, we observed a significant decrease in TE

for RNAiMAX (N/P 2) and even a total loss in TE for DOTAP:DOPE mRNA-LPXs. Several studies

suggest that aggregation and dissociation of the LPXs as well as an alteration of their surface

charge upon interactions with serum proteins decreases their TE in serum. (Dewitte et al.,

2014b; Zelphati et al., 1998; Zhang and Anchordoquy, 2004) Possibly due to nucleic acid

degradation (due to dissociation of the LPXs), a diminished interaction with the cell surface

(due to an altered surface charge) and a decreased internalization of the LPXs. (Zhang and

Anchordoquy, 2004) As a plus, there has been suggested that serum proteins could

41

negatively interfere with the ability of DOPE to induce endosomal release. (Zhang and

Anchordoquy, 2004)

Interestingly, when DOPE was replaced by high contents of cholesterol, the

transfection efficiency of the corresponding mRNA-LPXs (i.e. DOTAP:CHOL, N/P 2.5) did not

significantly alter in the presence of serum (5% FCI). Similar observations were obtained in a

study of Crook et al. and Zhang et al. In these studies, inclusion of cholesterol into

DOTAP/DNA LPXs resulted in a significant enhancement of transfection in the presence of

serum. (Crook et al., 1998; Zhang and Anchordoquy, 2004) Whether a diminished binding of

serum proteins to cholesterol containing LPXs could explain the enhanced transfection is a

recurring topic of discussion. (Li et al., 1999; Pozzi et al., 2012; Zhang and Anchordoquy,

2004) Zhang et al. and Betker et al. demonstrated that the presence of phase separated

cholesterol, otherwise referred to as cholesterol domains, improves resistance to serum-

induced aggregation and dissociation which could explain the improved transfection

efficiency. (Betker et al., 2013; Zhang and Anchordoquy, 2004) In this study, we proved that,

for both DOTAP:DOPE and DOTAP:CHOL mRNA-LPXs, full mRNA-incorporation remained in

the presence of serum. Thus, a difference in premature mRNA-release upon interaction with

serum components could not explain our observations. In order to assess whether an

improved resistance to aggregation could be observed for the DOTAP:CHOL mRNA-LPXs used

in our study, single particle tracking (SPT) experiments could be performed in serum-

containing medium.

Recently, Pozzi et al. investigated the uptake and intracellular trafficking of both DC-

Chol/DOPE DNA LPXs and DC-Chol-cholesterol/DNA LPXs by Chinese hamster ovary (CHO)

cells. The rationale for this study was to provide novel insights into the enhanced TE in

serum upon cholesterol incorporation. Remarkably, the experiments in this study were

performed in serum-free medium. The results indicate that DC-Chol-DOPE/DNA LPXs were

mainly taken up by the macropinocytosis pathway. However, upon replacement of DOPE by

cholesterol, a decreased colocalization with macropinosomes and lysosomes was observed.

The authors suggest that cholesterol-containing LPXs are able to efficiently escape from

macropinosomes. Furthermore, the authors demonstrate an increased contribution of a

vesicle-independent uptake process upon replacement of DOPE by cholesterol which could

also explain the reduced colocalization with macropinosomes. (Pozzi et al., 2012) To verify

42

whether similar differences in uptake and intracellular trafficking can be observed for the

particles and cells used in our study, a fluorescence-based colocalization assay could be

used. Furthermore, to assess the impact of serum on the cellular uptake and intracellular

trafficking of both DOTAP:DOPE and DOTAP:CHOL mRNA/LPXs, this assay could be

performed in both serum-free and serum containing medium. In this way, we can verify

whether differences between the two LPXs, in terms of cellular processing in the presence of

serum, could explain the observed differences in TE.

Taking their superior capacity to induce transfection in serum-containing medium

into account, we decided to continue further experiments with DOTAP:CHOL mRNA-LPXs at

N/P 2.5. During further experiments, we evaluated whether unmodified DOTAP:CHOL

mRNA-LPXs as such were capable of inducing DC-maturation. In fact, besides efficiently

introducing TAAs, an optimal lipid-based mRNA delivery system has to stimulate a full

maturation of the TAA-loaded DCs. We were able to show that unmodified DOTAP:CHOL DC-

loading resulted in a very small but significant upregulation of CD40 maturation marker-

expression. This very small shift in DC-maturation status is most likely induced by the

cationic lipid DOTAP within the mRNA-LPXs. In fact, Vasievich et al. illustrated that DOTAP is

capable of inducing an upregulation of co-stimulatory molecules. (Vasievich et al., 2011) This

could also be observed in a study of Yan et al. Interestingly, in this study, the authors point

out the role of a DOTAP-induced generation of reactive oxygen species (ROS) in the

enhanced maturation marker expression. (Yan et al., 2008) However, unlike the results

obtained in the above mentioned studies, we could not detect a significant upregulation of

CD86-expression and cytokine secretion. Two discrepancies in study design might explain

these differences. First of all, the BM-DCs in our study were exposes to much lower amounts

of DOTAP as compared to the above mentioned studies. Secondly, we used mRNA-LPXs

composed of DOTAP and cholesterol. Possibly, cholesterol could alter the effect of DOTAP on

the DC-maturation marker expression and cytokine secretion. In conclusion, the unmodified

DOTAP:CHOL mRNA-LPXs used in our study could only induce limited DC maturation.

Nevertheless, the limited DC maturation induced by unmodified DOTAP:CHOL mRNA-

LPXs already enabled DCs to induce extensive CD8+ T cell proliferation. The same could be

observed for DOTAP:DOPE:DSPE-PEG-2000-biotin mRNA-LPXs upon DC sonoporation by

Dewitte et al. (Dewitte et al., 2014) However, at a first glance, the quality of CD8+ T cells in

43

terms of cytolytic activity does not seem to be very high. For that reason, the added value of

different adjuvants (i.e. MPLA, CpG ODN, TriMix mRNA) was examined. With regard to the

generation of T cell immune responses, several studies have already demonstrated that

concomitant delivery of TAAs and adjuvants is optimal. (Silva et al., 2013; Zhang et al., 2007)

Therefore, we co-encapsulated the different adjuvants into the DOTAP:CHOL mRNA-LPXs.

First of all, we evaluated whether the co-incorporation of the different adjuvants into

the DOTAP:CHOL mRNA-LPXs could influence the transfection efficiency. In fact, Van Lint et

al. observed a reduction in protein expression when DCs were pulsed with Fluc mRNA in the

presence of LPS and MPLA. (Van Lint et al., 2012) Moreover, a hampered internalization of

naked mRNA (i.e. dependent on macropinocytosis) and an inhibition of mRNA cap-

dependent translation upon DC maturation is already described. (Diken et al., 2011; Lelouard

et al., 2007)

In this study, a decrease in TE was observed for TriMix containing DOTAP:CHOL

mRNA-LPXs. The same could be observed by Dewitte et al. (Dewitte et al., 2014) However, in

this specific case, the reduction in transfection efficiency was not unexpected as we were

forced to use a lower amount of GFP encoding mRNA (0.5µg instead of 1µg) due to toxicity

limitations. Furthermore, Dewitte et al. suggested that competition for mRNA-translation

upon co-delivery of multiple RNAs could also partly be accountable for the diminished

percentage of GFP-expressing DCs. This hypothesis was supported by observations of Chen

et al. and Bonehill et al. (Bonehill et al., 2008; Chen et al., 2013) Interestingly, we could not

observe any decrease in transfection efficiency upon co-incorporation of MPLA or CpG ODN

into the DOTAP:CHOL mRNA-LPXs. These results are in contradiction with the results

observed by Van Lint et al. (Van Lint et al., 2012) A possible explanation for this discrepancy,

is the difference in kinetics upon co-delivery of adjuvant and protein-encoding mRNA. We

can only hypothesize, that mRNA transfection is less hampered when the mRNA

transfection- and DC activation-process are synchronized. (Pollard et al., 2013) Another

conceivable explanation is a difference in the internalization pathways between naked

mRNA and DOTAP:CHOL mRNA-LPXs. In fact, Platt et al. elegantly illustrated that certain

forms of endocytosis (i.e. macropinocytosis and phagocytosis) are down-regulated upon DC

maturation, while others are unaffected by the maturation process.

44

With regard to DC activation, we show a significant enhancement in CD40 and CD86

maturation marker expression upon DC-loading with all types of immunomodulatory

DOTAP:CHOL mRNA-LPXs. Moreover, the CD40 and CD86 upregulations induced by CpG ODN

(i.e. TLR9 agonist) and MPLA (i.e. TLR4 agonist) containing DOTAP:CHOL mRNA-LPXs were

nearly similar to these observed for LPS (i.e. TLR4 agonist) stimulated DCs. Additionally, a

significant increase in both IL-12p70 and IL-10 could be demonstrated for both CpG ODN and

MPLA containing mRNA-LPXs. Moreover, with regard to the ratio IL-12p70 to IL-10, CpG ODN

was superior to LPS. Observed differences in cytokine secretions upon addition of the

different TLR-ligands could possibly be due to differences in signaling pathways. (Dowling et

al., 2008; Silva et al., 2013) Notably, the influence of TriMix on the DC maturation status is

only limited as compared to the LPS stimulated positive control group. Similar observations

were seen by Dewitte et al. However, these findings are in contradiction with the

observations of Bonehill et al. In this study, a marked enhancement of maturation marker

expression as well as an increased secretion of cytokines could be observed upon DC

electroporation with TriMix mRNA. (Bonehill et al., 2008) A possible explanation for the

observed discrepancies is the lower amount of TriMix mRNA used in our study. In the study

of Bonehill et al. DCs were electroporated with 10µg of each mRNA sequence. (Bonehill et

al., 2008) In contrast, due to toxicity limitations with the LPXs, we had to reduce the quantity

of each individual mRNA sequence to 0.5µg. Another possible explanation could be the

difference in transfection efficiency attained with DOTAP:CHOL lipofection and mRNA

electroporation. Indeed, in comparison to the 80-90% transfection obtained with mRNA-

electroporation, the percentage of GFP-expressing DCs upon DOTAP:DOPE mRNA lipofection

is rather low. (Dewitte et al., 2014a; Michiels et al., 2005)

Taking the transfection efficiency and the effects on DC maturation status into

account, the developed immunomodulatory DOTAP:CHOL mRNA-LPXs appear to be superior

to the unmodified DOTAP:CHOL mRNA-LPXs. Ultimately, we examined whether this

translates into superior T cell proliferation and CTL responses. With regard to T cell

proliferation, we could not yet detect a significant difference between unmodified and

immunomodulatory DOTAP:CHOL mRNA-LPXs. However, we could detect a trend towards

more extensive T cell proliferation upon inclusion of adjuvant. Moreover, antigen-specific

lysis as a result of CTL responses seems to increase when adjuvants are co-incorporated into

45

the DOTAP:CHOL mRNA-LPXs. However, with regard to both T cell proliferation and CTL

responses, we could not yet observe significant differences between the different

immunomodulatory mRNA-LPXs. However, we must point out the necessity to repeat these

experiments to confirm the obtained results before we can draw final conclusions.

In conclusion, we were able to design a serum-stable lipid-based delivery tool that

stimulates DCs in vitro to induce potent cytotoxic T cell responses against specific TAAs. The

development of such a tool is a major step forward towards developing in vivo applicable

DC-vaccines.

46

6. CONCLUSION

In this work, our main challenge was the development of a serum-stable particulate

delivery system that is able to efficiently deliver TAAs into BM-DCs in vitro. Different lipid-

based delivery tools were characterized and their capacity to introduce mRNA encoded

proteins into BM-DCs in vitro, both in serum-free and serum-containing medium, was

evaluated. Of all particles, the highest transfection efficiency in the presence of serum could

be obtained with DOTAP:CHOL mRNA-LPXs (N/P 2.5). The prepared DOTAP:CHOL mRNA-

LPXs exhibited a mean particle size of 154.0 ± 0.8 nm and a zeta potential of 47.2 ± 1.8 mV.

Furthermore, complete mRNA-incorporation at the used N/P ratio, both in serum-free and

serum-containing medium, could be verified by means of agarose gel electrophoresis. The

exact mechanism accountable for the enhanced TE observed upon replacement of DOPE by

cholesterol in DOTAP:DOPE LPXs is yet to be determined.

In a second phase of this study, the impact of unmodified DOTAP:CHOL mRNA-LPX

loading on the DC-maturation status was determined by evaluating both the expression of

DC maturation markers and the DC-cytokine secretion. In fact, besides efficiently introducing

TAAs, an optimal lipid-based mRNA delivery system has to stimulate full DC-maturation. The

results showed that unmodified DOTAP:CHOL mRNA-LPXs as such, could only induce a very

small, but significant shift in CD40 expression. In contrast, no increase in CD86 expression

and cytokine secretion could be observed. These results indicate only limited DC maturation

upon DC-loading with unmodified DOTAP:CHOL mRNA-LPXs. Furthermore, it was shown that

the capacity of DCs to respond to the well-known maturation-stimulus LPS was not

negatively influenced by particle loading. These observations prompted us to include

additional adjuvants into the mRNA-LPXs to further increase the DC maturation-status.

The impact of the co-delivery of three different adjuvants, namely CpG ODN, MPLA or

TriMix mRNA on the DC-maturation status was evaluated in a flow cytometry experiment.

Extensive upregulation of CD40 and CD86 expression as well as an increase in cytokine

secretion could be observed for both CpG ODN and MPLA. This proves that both adjuvants

can efficiently promote DC maturation. Moreover, no decrease in transfection efficiency was

detected upon co-delivery of these adjuvants. Furthermore, although a significant

upregulation of CD40 and CD86 was observed with TriMix containing mRNA-LPXs, the

contribution of TriMix mRNA was only limited as compared to the above mentioned

47

adjuvants and no increase in cytokine secretion could be detected. Moreover, an expected

decrease in transfection efficiency was observed.

Ultimately, we evaluated the capacity of unmodified and immunomodulatory (i.e.

containing adjuvants) mRNA-LPXs to stimulate DCs in vitro to trigger CD8+ T cell proliferation

and cytotoxic T cell responses, which was evaluated by means of an in vitro T cell

proliferation assay and a CTL assay respectively. With regard to T cell proliferation, we

proved that extensive T cell proliferation was triggered by both DCs loaded with unmodified

and immunomodulatory DOTAP:CHOL mRNA-LPXs. Interestingly, a much lower T cell

proliferation could be observed for DOTAP:DOPE mRNA-LPX loaded DCs which points out the

superiority of a serum resistant carrier. However, although a trend was detected towards

more extensive T cell proliferation upon addition of adjuvant, we could not yet prove

significant differences between the unmodified and the different immunomodulatory

DOTAP:CHOL mRNA-LPXs. With regard to antigen-specific CTL responses, we proved that co-

incorporation of adjuvants results in increased CTL responses. However, we must point out

the necessity to repeat these experiments before drawing final conclusions. We

demonstrated a serum-stable lipid-based delivery tool that stimulates DCs in vitro to induce

potent cytotoxic T cell responses against specific TAAs. The development of such a tool is a

major step forward towards developing in vivo applicable DC-vaccines.

48

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