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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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