FACHBEREICH BIOLOGIE
Antigenbeladung von Dendritischen Zellen mit PLGA-Mikrosphären zur
Immuntherapie bei Tumoren
Dissertation
Zur Erlangung des akademischen Grades eines
"Doktors der Naturwissenschaften"
(Dr. rer. nat.)
des Fachbereiches für Biologie an der Universität Konstanz
vorgelegt von Eva Christine Schlosser
Tag der mündlichen Prüfung: 12.12.2007 1. Referent: Prof. Marcus Groettrup 2. Referent: Prof. Marcel Leist
Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/4645/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-46456
Table of contents
I
Abreviations IV
Zusammenfassung/ summary VI
Chapter 1 Introduction: 1
1. The principal mechanism of immune response 2 2. Dendritic cells 3 3. Tumor immunotherapy 4 4. Dendritic cells in cancer immunotherapy 6
5. Adjuvants 8
6. Immunostimulatory adjuvants 9 7. Delivery devices 11 8. PLGA particles as an antigen delivery device 13 9. References 15
Chapter 2 Encapsulation of proteins and peptides into biodegradable 19 Poly (D,L-lactide-co-glycolide) microspheres prolongs
and enhances antigen presentation by human dendritic cells
1. Abstract 20
2. Introduction 20 3. Materials and Methods 21
4. Results 25
5. Discussion 28
6. References 29
Chapter 3 Ex vivo loading of dendritic cells with peptide loaded PLGA 31 Microspheres enhances T-lymphocyte response
1. Abstract 32
2. Introduction 32
3. Results 33
4. Discussion 39
5. Materials and methods 41 6. References 43
Chapter 4 The encapsulation of maturation stimuli increases the immune 46 response after direct injection of PLGA-microspheres
1. Abstract 47
2. Introduction 47
3. Results 49
4. Discussion 54
5. Materials and methods 57
6. References 59
Chapter 5 Preparation and analysis of PLGA-microspheres 61
1. Abstract 62
2. Introduction 62
3. Results 65
4. Discussion 69
5. Materials and methods 72
6. References 74 Chapter 6 TLR ligands and antigen need to be coencapsulated into the same 77
biodegradable microsphere for the generation of potent cytotoxic T lymphocyte responses
1. Abstract 78
2. Introduction 78
3. Results 80
4. Discussion 90
5. Materials and methods 94
6. References 97 Chapter 7. in vitro cross presentation of PLGA microspheres 101
1. Abstract 102 2. Introduction 102 3. Results 105
4. Discussion 109
5. Materials and methods 112
6. References 113 Chapter 8: Evaluation of the capacity of PLGA microspheres to 117
induce immune responses sufficient to eradicate tumour growth
1. Abstract 118 2. Introduction 118
3. Results 119
4. Discussion 124
5. Materials and methods 127
6. References 128 Chapter 9: conclusion and outlook 131
1. Abstract 140 2. Introduction 140 3. Results and discussion 141 5. Materials and methods 145
6. References 145
II. References 146
III. Record of achievement/ Eigenabgrenzung 162
IV. List of publications 163
V. Acknowledgements 164
List of abbreviations
Aa amino acids
APC antigen presenting cells
BMDC bone marrow derived dendritic cell BSA bovine serum albumin
CCL CC chemokine ligand CCR CC chemokine receptor CD cluster of differentiation
CpG cytosin-phosphatidyl-guanosin CTL cytotoxic t-lymphocytes
DC dendritic cell
DMEM Dulbecco`s modified eagle medium DMSO Diemethylsulfoxid
DNA deoxyribonucleic acid
EDTA Ethylenediamine-tetraacetic acid ER endoplasmic reticulum
FACS fluorescence activated cell sorting FCS fetal calf serum
FDA food and drug administration FITC Fluorescein isothiocyanat
GMCSF granulocyte macrophage colony stimulating factor GST glutathione-S- transferase influenza virus matrix HA Influena haemagglutinin protein
HLA human leucocyte antigen i.v. intravenously
IFA incompletes Freund`s Adjuvans IFN interferon
Ig immunoglobulin IL interleukin
IMDM Iscove´s modified dubelccos´s medium kDa Kilo dalton
LPS lipopolysaccharide
MHC major histocompatibility complexes
NK cells natural killer cells
NOD like nucleotide binding oligomerization domain-like receptors PAGE polyacrylamide gel electrophoresis
PAMP pathogen associated molecular pattern PBMC peripheral blood mononuclear cells PBS phosphate buffer saline
PFA Paraformaldehyde Pfu plaque forming units PGE2 Prostaglandin E2
PLGA-MS Poly (D,L-lactic-co-gycolic acid) microsphere poly I:C polymer of inosinic and cytidylic acid
PRR pathogen recognition receptor PSCA prostate stem cell antigen RNA ribonucleic acid
s.c. subcutanously T regs regulatory T-cells
TAA tumour associated antigen TCR T-cell receptor
TGF transforming growth factor Th T helper cell
TIL tumour infiltrating lymphocyte TLR toll like receptors
TNF tumour necrosis factor v/v volume per volume VLP virus like particle w/v weight per volume
µm micrometer
µM micromolar
µm micro meter
Summary
The WHO reported in 2005 that cancer accounts for 13% of all deaths; therefore cancer is still the leading cause for death worldwide. For a successful cancer treatment, complete removal of the cancerous cells without causing damage to the rest of the body is the ultimate goal. Besides the conventional therapies, such as surgery, chemotherapy and radiation, specific activation of the immune system represents a new and promising approach which might meet the above mentioned criteria very well. The use of dendritic cells as cellular vaccines represents one way to program the immune system in such a way that it recognizes and consequently eliminates selectively tumour cells: Dendritic cells can be isolated from patients and pulsed ex vivo with peptides which are exclusively expressed on tumour tissue. Then, these cells are re-injected into the patient, where they licence CD8+ lymphocytes to eliminate selectively the tumour cells.
In this study a vaccination regime based on PLGA- microspheres as a carrier system to deliver antigens towards antigen presenting cells in vivo is established, optimized and investigated on its efficacy in a mouse model.
In the first two chapters we show that immunisation with dendritic cells externally loaded with PLGA-MS is superior to the conventional immunisation method using externally peptide pulsed dendritic cells. Nevertheless, if PLGA-MS loaded with antigen are injected directly into the animal, the response is rather weak but co- administration of maturation stimuli is able to rescue the response. Additionally not only encapsulation of antigen or adjuvant but also the delivery of antigen and adjuvant in close proximity -as it can be provided by the PLGA-MS system- leads to a significantly increased CD8+ lymphocyte responses.
Because efficient cross presentation is a prerequisite for the induction of a potent immune response a detailed investigation of the cross presentation pathway of PLGA-MS in vitro is performed in chapter 7. Finally, the capacity to protect and cure animals from the development of cancer was assessed in the last chapter.
Taken together this study not only shows some mechanistical aspects which should be kept in mind when vaccination strategies are designed, it also paves the way for a first clinical trial using PLGA-MS as an antigen carrier system.
2005 berichtete die WHO, Krebs stelle mit weltweit 13% immer noch die Todesursache Nummer eins dar. Das Ziel einer erfolgreichen Krebstherapie ist die komplette Entfernung aller Tumorzellen, ohne dabei das umliegende gesunde Gewebe zu zerstören. Neben den konventionellen Therapieformen, wie operativen Maßnahmen, Chemotherapie und Bestrahlung stellt die Immuntherapie einen neuen viel versprechenden Ansatz in der Krebsbekämpfung dar.
Der Einsatz von dendritischen Zellen - mit Tumorantigenen beladen- als zellulärer Impfstoff ist ein Weg, das Immunsystem so zu „programmieren“ dass es alleinig Tumorzellen als „fremd“ erkennt und anschließend eliminiert. Hierfür werden dendritische Zellen aus dem Patienten isoliert und ex vivo mit ausschließlich auf Tumoren exprimierten Peptiden beladen. Dieser Zellen können dann nach Rückinjektion in den Patienten CD8+ Lymphozyten derart aktivieren, dass sie selektiv das Tumorgewebe zerstören.
In der vorliegenden Studie wurde ein Impfsystem basierend auf PLGA-Microsphären, welche dazu dienen als Trägersystem in vivo antigenpräsentierende Zellen mit Antigenen zu beladen, aufgebaut, optimiert und seine Effizienz in der Maus als Tiermodel untersucht.
In den ersten zwei Kapiteln konnte gezeigt werden, dass Immunisierung mit dendritischen Zellen, die ex vivo mit PLGA-Microsphären beladen wurden zu stärkeren Immunantworten führte als die konventionelle Therapie basierend auf Peptid gepulsten dendritischen Zellen. Nichtsdestotrotz, nach direkter Injektion von PLGA-Microsphären war die Aktivierung der CD8+-Lymphozyten nur sehr schwach, konnte jedoch durch Verabreichung eines Adjuvants immens verstärkt werden.
Wir konnten außerdem zeigen, dass sich die Verkapselung von Antigen und Adjuvantien in einer erhöhten CD8+-Lymphozyten Aktivierung widerspiegelt. Vor allem aber führte die Darreichung von Antigen und Adjuvant in enger räumlicher Nähe, wie sie durch Coverkapselung in PLGA-Mikrosphären erreicht wird, zu einer signifikant gesteigerten Immunantwort.
Eine Grundvoraussetzung für das Auslösen einer wirksamen Immunantwort ist eine effiziente Antigenverarbeitung in der Zelle über die sog. „Kreuzpräsentierung“. Daher wurde in Kapitel 7 eine detaillierte Studie des Verarbeitungsweges von PLGA-
PLGA-Mikrosphären-Impfsystems sowohl in Hinsicht auf die Behandlung vorhandener Tumore als auch in Hinsicht auf den Schutz vor der Entstehung eines Tumors im Tiermodell gezeigt.
Zusammenfassend wurden in dieser Studie zum einen generelle mechanistische Aspekte die bei dem Design von neuen Impfsystemen berücksichtigt werden sollen gezeigt; zum anderen auch die Wirksamkeit des PLGA-Mikrosphärensystems im Tiermodel Maus bewiesen, welches einen wichtigen Schritt für erste klinische Studien darstellt.
Chapter 1
Introduction
1.The principal mechanisms of immune responses
The vertebrate immune system possesses a variety of defense mechanisms against invading pathogens. Immediately after infection the innate immune system provides the first line of defense against invading pathogens. The innate immunity is characterized by unspecific mechanisms such as physical and chemical barriers (for example mucous membranes, tight junctions in the skin, secretion of defensines and lysozyme) as well as activation of the complement system, release of inflammatory molecules (i.e. Chemokines, Eicosanoids and cytokines) and the activation of phagocytes and natural killer (NK) cells. The response of the innate immunity is characterised by being immediate and unspecific and does not lead to a memory.
The receptors involved in innate immunity such as toll like receptors (TLRs), Mannose receptors, complement receptors and scavenger receptors are all germline encoded and are found in nearly all forms of life (reviewed by Ezekowitz et al., 1996) Innate immunity controls the infection until the delayed adaptive immune response takes place. The adaptive immune response has developed early in vertebrates and allows for a stronger immune response as well as for immunological memory. The adaptive immunity can be subdivided in humoral and cell-mediated responses.
The humoral response is mediated by antibody secreting B-cells, while the cell- mediated response is represented by T-helper cells and cytotoxic T-lymphocytes (CTLs). T-cells carry highly specific receptors, which recognize the antigen exclusively if it is processed and subsequently presented on major histocompatibility complexes (MHCs) by professional antigen presenting cells (APC). These cells specialised in antigen uptake, processing and presentation, are B-cells, macrophages and most importantly the highly specialised dendritic cells (DCs).
CTLs carry the CD8 coreceptor and recognize antigens presented on MHC-I molecules through their T-cell receptor (TCR), while T helper cells are characterized by a CD4 coreceptor and are MHC-II restricted. When CTLs are activated they migrate towards the site of infection, where they recognize the antigen presented on MHC-I of infected cells and eliminate these through perforin and granzyme release (Kagi et al., 1994). T-helper cells are not able to kill target cells. Their function is rather to induce and modulate the immune response through cytokine release (Swain et al., 1983). The group of T-helper cells is subdivided into Th1, Th2 cells and Th17 cells. Th1 cells secrete IFN-γ and mediate killing by CTLs whereas Th2 cells secrete
A humoral immune response is generally required for the eradication of extracellular pathogens, while a cellural immune response is needed for combating intracellular pathogens and tumour cells.
2. Dendritic cells
Dendritic cells have been first described by Paul Langerhans in 1868 but thought to be cutaneus nerve ends. Steinman and Cohn finally identified these cells 1973 as part of the immune system (Steinman et al., 1973).
Dendritic cells are widely accepted as the most potent APCs, and are hence called
“sentinels” of the immune system. They play a crucial role in inducing protective adaptive immune responses against foreign as well as tolerance to self antigens.
In their immature state they are continuously patrolling the body and thereby sampling the environment. For this purpose they are especially equipped with a variety of uptake receptors, namely mannose receptor, DC-Sign, DEC 205, scavenger receptor and Fc receptors (all reviewed in Proudfoot et al., 2007). The incorporated antigens are then processed and finally presented on surface MHC-I or MHC-II molecules. If dendritic cells capture pathogen encoded antigens in context of an inflammatory stimulus they are activated in the periphery via maturation which is the essential step for enabling dendritic cells to migrate towards the draining lymph node, where they activate naïve T-cells that recognize the presented antigens (Reis e Sousa et al., 2001).
Inflammatory stimuli are represented by highly conserved pathogen associated molecular patterns (PAMPS). Maturation of dendritic cells is mainly mediated through the recognition of these motifs by Toll like receptors (TLRs), highly expressed on immature dendritic cells (Akira et al., 2006) or other recently identified receptors as the nucleotide binding oligomerization domain-like receptors (NOD-like) (Kaparakis et al., 2007).
As an important consequence of maturation, dendritic cells acquire the capacity to migrate towards lymph nodes via upregulation of the chemokine receptor CCR7
connected with increased responsiveness to the chemokines CCL19 and CCL21, which are chemoattractants produced in the draining lymph node (Sallusto et al., 1998). Maturation is also associated with upregulation of molecules for antigen presentation, such as MHC-I and MHC-II molecules as well as the costimulatory molecules CD80 and CD86, which are also essential for proper priming of CD8+ T- cells.
When dendritic cells have reached the lymph node they undergo another process termed “licensing” which is mediated via cross linking of CD40 on the DCs with CD40L expressed on antigen activated CD4+ helper cells (Schoenberger et al., 1998). Licensing is a prerequisite for further upregulation of costimulatory molecules, for instance OX-40 L and 4-1BBL. It also triggers the production of IL-12, a cytokine which drives the polarization of the T-helper cell response towards a Th1 phenotype required for the efficient activation of CTLs.
Unlike mature DCs, immature Dendritic cells are not only unable to stimulate T-cells, they also take part in the immune surveillance by inducing peripheral T-cell tolerance.
This has been shown in the elegant studies by Probst et al., 2005 using a mouse model in which antigens were presented by immature dendritic cells leading to CD8 T-cell tolerance via a T-cell intrinsic mechanism. A T-cell extrinsic mechanism in induction of peripheral tolerance mediated by iDCs is the activation of regulatory T- cells (T regs) (McIlroy et al., 2003).
3. Tumor Immunotherapy
The concept of tumor surveillance by Burnet suggested that the immune system is able to recognize tumour cells as “nonself” (Burnet, 1970). Nowadays it is known that this is mainly true for virally transformed tumour cells with the exception for some cases of melanoma and renal tumour cells.
Whether this is due to a process called immunoediting which assumes that a CTL mediated anti tumour immune response exists, but fails because tumours have managed to escape through several mechanisms (Swann et al., 2007) or whether most tumours simply do not induce an immune response (Klein et al., 2005) is still a matter of debate.
immunosurveillance aiming at the eradication of tumour cells were developed.
Indeed, although vaccination against infection has a major impact on worldwide public health, development of cancer vaccines is more difficult. Most challenging in the development of cancer vaccines is the need to break immune tolerance against a self antigen to induce a tumour specific cellular immune response strong enough to induce tumour cell destruction.
It is generally accepted that CTLs and T helper cells are both required to mount a vigorous cellular immune response against tumours. Therefore the two main strategies in immunotherapy are the parenteral administration of dendritic cells charged with the respective antigens for induction of CTL responses or the adoptive transfer of autologous in vitro activated T-cells.
Therapy using autologous T-cells is based on transfusion of either autologous lymphocytes restimulated with tumour associated epitopes in vitro, in vitro activated and expanded tumour infiltrating T-lymphocytes (TIL), or T-cells with genetically engineered TCR specific for tumour associated antigens (TAAs) (reviewed in June, 2007). For adoptive transfer with TILs a tumour regression in 50% of all lymphodepleted patients has been shown in a study by Dudley et al., 2002. Despite these very encouraging results, ex vivo activation and expansion of T cells is very laborious and expensive and has to be “personalized” for every patient.
Another promising approach for the in vivo activation of CTLs for tumour treatment is the blockade of inhibitory checkpoints on T-cells with antibodies:
CTLA-4 and PD-1 are immunoregulatory molecules, expressed on activated T cells capable of down-regulating T cell activation, termed “functional exhaustion”
(Freeman et al., 2000). Therefore they might represent good targets for antibody mediated immune modulation. For example, the usage of antagonistic antibodies for CTLA-4 in combination with IL-2 led to 22% objective tumour regression in melanoma patients (Maker et al., 2005). Because it was shown that the ligand for PD-1 is expressed on mouse melanoma cells and specific blocking of PD1-ligand with an antibody inhibited tumour cell growth (Iwai et al., 2002), PD-1 might represent another suitable target. Current studies using PD-1 antibodies in clinical trials are ongoing. Admittedly blockade of inhibitory molecules might always be connected with the induction of autoimmune responses causing a lot of side effects.
The largest and maybe most successful field in immunotherapy is still the use of monoclonal antibodies for treatment of cancer. In 2006 there were 12 monoclonal antibodies already approved and further 160 different antibodies in clinical trials or waiting for approval by the FDA. Monoclonal antibodies are used for specific targeting of tumour cells. Rituximab, specific for CD20 specifically expressed on B- cells, was the first one approved to treat malignancy (non Hodgkin lymphoma).
Another prominent candidate is Herceptin, which targets HER2/neu expressing breast cancer cells. These antibodies work by antibody –dependent cellular cytotoxicity, direct cytotoxic activity and inhibiton of receptor signalling (Zafir-Lavie et al., 2007).
4. Dendritic cells in cancer immunotherapy
One approach to induce an immune response against cancer is the immunization of patients with their own dendritic cells loaded ex vivo with antigens expressed exclusively on the tumour, so called tumour associated antigens (TAAs).
The first promising DC vaccination study in which autologous dendritic cells were externally loaded with host idiotype protein and reinjected in B-cell lymphoma patients was already published in 1996 by Hsu et al.. Meanwhile a substantial number of clinical trials using dendritic cells as a vaccine against more than 20 different types of tumours has been carried out over the last decade (list of recently published trials available at http://www.mmri.mater. org. au/).
Although tumour specific immune responses have been frequently observed, durable clinical responses were rare, leading to the conclusion that although DC vaccination has proved to be very safe further optimization is needed.
Critical points in establishing a potent immunization approach using dendritic cells is the large scale generation of mature dendritic cells in vitro as well as an optimal in vitro antigen loading strategy.
There exist several attempts of loading strategies including different possibilities for antigen sources: Epitopes, tumour associated proteins or even whole tumour cell material can be used as well as nucleic acid abased loading approaches like antigen encoding DNA or RNA.
multiple epitopes. Unfortunately this approach has been hampered by low transfection efficiencies and therefore suitable loading strategies have to be developed.
The „classical way” of external loading of dendritic cells with synthetic epitopes exhibits several drawbacks such as MHC-restriction, which confines immunotherapy to patients expressing the appropriate restriction element and the need for identification of epitopes, which is especially important for CD4+ helper epitopes which are rarely known (Velders et al., 2003). Therefore also T-cell help is missing if dendritic cells are externally loaded with peptides. Another obstacle is represented by the rapid turn over of class I-peptide complexes on the cell surface (Ludewig et al., 2001; Dieckmann et al., 2005; Kukutsch et al., 2000), which leads to a limited duration of T cell stimulation which in turn hampers successful priming of T-cells (Spierings et al., 2006).
Despite using peptides as antigen source, proteins offer the benefit of potentially containing multiple epitopes, including class II helper epitopes. Use of proteins as antigen source also avoids the need for HLA restriction. Otherwise large scale production of proteins for clinical use is much more difficult than synthesis of peptides. Moreover, in order to be presented on class I, proteins must undergo a process called cross processing, which means uptake and delivery into the MHC-I cross presentation pathway, which has been reported to be very inefficient in vivo (Wolkers et al., 2004). Therefore, loading of dendritic cells with proteins seems to be suitable, but requires delivery by a carrier system.
Taken together, to circumvent the very complicated and expensive Dendritic cell reinjection strategy the approach of a system which delivers proteins in vivo into the MHC-I pathway would be very convenient.
5. Adjuvants
Because of the general problem, that vaccines alone are often poorly immunogenic there is an urgent need for the development of potent and safe substances for improving immunogenicity. Such agents, termed adjuvants were first recognized by Ramon in 1925 and described as “substances which used in combination with a specific antigen produce a more robust immune response than the antigen alone”
The first reported use of adjuvants was in 1926 when Glenny demonstrated that aluminium compounds could enhance the immunogenicity of a diphtheria toxoid vaccine. Since then the only adjuvants currently approved by the US food and drug administration (FDA) remain aluminium based mineral salts. Despite their reported good safety record aluminium salts bias the immune response toward Th2 and thereof lead to a diminished Th1 response. Moreover alumn adjuvants have also been associated with increased levels of IgE connected with allergic reactions in some individuals (Gupta et al., 1998). Thus, there is an urgent need for the development of safe and effective adjuvants.
Adjuvants can be classified into two classes, based on their principal modes of action: a) immunostimulatory adjuvants, and b) vaccine delivery systems.
T-cell stimulation needs basically two different signals. The first signal is provided by the peptide which is presented on MHC complexes towards the TCR. Adjuvants based on vaccine delivery systems significantly increase delivery of antigens into the MHC-I presentation pathway, thus increasing signal 1. Immunostimulatory adjuvants lead to dendritic cell maturation, meaning upregulation of costimulaotry molecules, which provides the essential second signal for the T-cells. In the absence of signal 2 immunological tolerance would even be induced.
Mostly used immunostimulatory adjuvants in cancer immunotherapy are: TLR- agonists and Cytokines. Cytokines are secreted low molecular weight proteins that modulate the duration as well as the intensity of the immune response.
Recombinant cytokines such as IL-2, TNFα and GMCSF have been used in cancer immunotherapy although their use has been limited due to their systemic toxicity. IL-2 for example is a glycoprotein that activates T-cells for clonal expansion and cytolytic activity. Clinical trials using an IL-2 regimen with/without lymphocyte activated killer cells in patients with metastatic melanoma and renal cell carcinoma showed 4-6%
complete response (Rosenberg et al., 1989). High-dose IL-2 received FDA approval in 1992 for the treatment of metastatic renal cell carcinoma and in 1998 for the treatment of metastatic malignant melanoma. Administration of IL-2 is connected with very strong side effects such as capillary leak syndrome leading to fluid retention, hypotension, adult respiratory distress syndrome, prerenal azotemia, and very rarely myocardial infarction (Kammula et al., 1998). Another disadvantage using IL-2 is the induction of immunosuppressive regulatory T-cells. Therefore the use of IL-7 or IL-15 as immunostimulators might be even more beneficial. Both cytokines are of importance in T-cell homeostasis and first studies suggest their efficacy in tumour treatment (Li et al., 2007; Waldmann, 2006)
It has been shown, that PAMPS have the ability to enhance the immune response by activating the APCs. PAMPS are recognized by several receptors like C-type Lectins, complement receptors or NODs (Geijtenbeek et al., 2004; Bajtay et al., 2006; (1).
The best characterized receptors among the pathogen recognition receptors (PRRs) are the Toll like receptors (TLRs). There are 11 different TLRs identified in mouse and men. They enable the immune system to recognize a wide range of pathogens.
TLRs are expressed on all APCs and are either located on the cell surface (TLR 1, 2, 4, 5, 6, 10 and 11) or within the endosomal compartments (TLR 3, 7, 8 and 9). Upon recognition of their ligands, TLRs transduce signals through two pathways involving distinct adaptor proteins.The adaptor MyD88 is utilized by all of the known TLRs except TLR3, which signals through TIR domain-containing adaptor molecule 1 (TICAM-1). TLR-4, the receptor for LPS, is signalling through either of the both
adaptor molecules. The signal cascade of all TLRs leads to activation of the nuclear factor κB (NF-κB) (reviewed by Iwasaki et al., 2004).
Activation of NFkB leads to:
• Upregulation of CCR7 and simultanously to downregulation of CCR5
• Upregulation of MHC-II complexes on the cell surface (Cella et al., 1997)
• Upregulation of costimulatory molecules CD80, CD86 and CD70 (Iwasaki et al., 2004)
• Improvement of cross priming in DCs and B-cells (Schroder et al., 2005;
Schulz et al., 2005; Heit et al., 2004)
• Biasing of the T helper reponse towards a Th1 response (Chu et al., 1997)
Therefore TLR ligands represent promising candidates for adjuvants in cancer immunotherapy.
This study focused on the use of two different TLR ligands as adjuvants, namely an unmethylated CpG motif (CpG 1826) and a polymer made of inosinic acid and cytidylic acid (poly I:C) .
Bacterial DNA which contains in contrast to human DNA unmethylated CpG motifs is the main stimulus for TLR9. In tumour vaccination approaches CpG increased the immunogenicity of peptide, DNA, whole tumour cells and DC based vaccines (van Duin et al., 2006). Numerous studies indicate that CpG Oligonucleotides enhances antibody responses as well as Th1 polarized T-cell responses to a wide variety of antigens even stronger than the gold standard comlete Freund´s adjuvant.
(reviewed in Klinman, 2004). Additionally, a first promising trial in which melanoma patients were vaccinated with a combination of CpG, incompletes Freund´s Adjuvans (IFA) and Melan A resulted in sustained immune responses in all of the patients (Speiser et al., 2005), suggesting that CpG Oligos might represent a useful tool in immunotherapy.
The second adjuvant used in this study was poly I:C. Poly I:C is a virus derived RNA motif and the ligand for it is the intracellular localized TLR-3. Signalling through TLR- 3 leads to interferon (IFN-α/-β) production, which mediates not only inhibition of viral replication (Tabeta et al., 2004), but also upregulation of MHC-molecules.
Additionally it promotes apoptosis through induction of transcription of p53. A fact
Another characteristic of poly I:C, namely the induction of cross presentation has been recently suggested by Schulz et al., 2005. In this study, poly I:C, when administrated in a cell associated form induced upregulation of costimulatory molecules. This study was supported by another one showing induction of CD8+ lymphocytes in vivo exclusively if poly I:C was administered in a cell associated manner (McBride et al., 2006).
7. Delivery devices
Apart from the immunostimulatory molecules mentioned above there exists a second way to improve immune reponses by delivering the antigen and the immunostimulatory substance to the APCs and concurrently stabilizing both. Delivery of a whole array of molecules has been the subject of intense research. As a consequence there is a large number of delivery systems available.
What are the requirements for an appropriate delivery approach used for tumour vaccination?
First of all such a carrier system should be able to increase the uptake by APCs, second it should be able to deliver the content into the MHC class I pathway. It should also increase the half life of the delivered substances (Mutwirin et al., 2004).
Last but not least it is suggested by several recent studies, that close proximity of antigen and adjuvant, provided by a carrier system leads to delivery not only to the same cell but also to one and the same endosome which might represent a prerequisite for efficient T-cell priming. (Blander et al., 2006; Wagner et al., 2004).
Examples for delivery vehicles which meet these criteria very well are:
1.) Virus like particles (VLPs) consist of viral proteins, but lack viral DNA and are therefore not infectious. It has been shown that they are taken up by dendritic cells and thereby inducing maturation. They enter the cross presentation pathway enabling an efficient CD8 T-cell response. A major
disadvantage of VLPs represents the fact that the carrier itself induces Ab responses, which might lead to rapid clearance of the carrier in a prime boost regimen (reviewed in Ramqvist et al., 2007).
2.) Liposomal carrier systems, which are spherical particles composed of phospholipids and cholesterol that self associate into bilayers encapsulating an aqueous interior. They are taken up by APCs and protect their content from rapid intracellular degradation. They also increase the MHC-I antigen presentation. Admittedly, some limitations of the use of liposomes has emerged, namely poor control over release, poor encapsulation efficiencies and instability during storage (Altin et al., 2006).
3.) Iscoms (Immune-stimulating complexes) are compared to liposomes much more stable delivery systems. They are made of Saponin, lipids and antigen.
They have a size of 40 nm diameter, which is comparable to viral size. They are also known for rapid incorporation by APCs and provide -although not complete- maturation of dendritic cells itself which is probably induced by the Saponin. A major hurdle in the usage of ISCOMS is the difficult particle preparation often connected with the requirement of extensive antigen modifications because mainly hydrophobic antigens can be packaged into the particles (reviewed in Sanders et al., 2005).
4.) chemical cross linking of antigen to CpG represents another mechanism which leads to increased immune responses. The close physical linkage of antigen and adjuvant presumably allows delivery of both components to one endosome, which is critical for the induction of an immune response. and might also increase the uptake rate by APCs. Unfortunately the procedure of chemical linkage can be very inefficient and varies between antigens (Heit et al., 2004; Tighe et al., 2000).
5.) live vectors as attenuated viruses: The first example for a recombinant viral vector system was vaccinia virus in 1982. Nowadays there are also other viral vectors in use, such as modified vaccinia virus type ancara, adenoviruses and Herpes simplex virus. Viruses drive antigen expression inside the cytoplasm of the cells they invade, thus there is no need for targeting the cross presentation
obtain. Another disadvantage of this method is the pre-existence of antibodies against the carrier which does not allow for booster immunisations. Moreover viruses contain many T-cell epitopes that will compete with the antigenic epitopes delivered (Harrop et al., 2006).
8. PLGA (Poly (D,L-lactic-co-glycolic acid) ) particles as an antigen delivery device
Strategies for loading dendritic cells in vivo with antigen are still a major topic in immunotherapeutic research. As a carrier system which might have the potential to solve all the aforementioned problems of conventional antigen delivery the PLGA microsphere system was used in this study.
PLGA is a polymer consistent of the two α-hydroxyacids, lactic acid and glycolic acid.
In aequeous solution the polymer hydrolysis in the two acids which can be metabolized via the citric acid cycle and is therefore completely biodegradable (Brady et al., 1973). This material is very well characterised and approved for application in humans by the FDA which represents an obvious advantage for an antigen delivery system (Singh et al., 2002; Johansen et al., 2000).
Fig.1: Chemical structure of PLGA polymer. The “m” component represents lactic acid, while the “n” component represents glycolic acid.
In contrast to most other studies microsphere production was performed in our study
solvent while the antigen is solved in water phase. Subsequently the two phases are emulsified. This dispersion is then atomised in a flow of drying air at slightly elevated temperature, thereby organic solvent is rapidly vaporised leaving behind solid microspheres that are separated from the drying air in a cyclone. This method is compared to the more commonly used evaporation processes very rapid, convenient and easy to scale up. It involves mild conditions and is less dependent on the solubility parameters of the drug and the polymer (different methods of encapsulation are reviewed by Jain et al., 2000). This method also provides the potential to adjust particles to any distinct size needed for any purpose.
For the 50% glycolic acid 50% lactic acid composition of the PLGA microspheres used in this study it has been reported that the polymer is hydrolyzed in aqueous media within a period of 20-30 days during which encapsulated proteins and peptides are released. Therefore PLGA microspheres provide continous delivery of the antigen source, which might overcome the above mentioned hurdle of rapid loss of MHC: peptide complexes observed when peptide pulsed dendritic cells were used for immunisation. The PLGA preparations yielded by spray drying have a size of 1-10 µM which represents the appropriate size for enhanced phagocytotic uptake by dendritic cells (Waeckerle-Men et al., 2005; Peyre et al., 2004). Another advantage of the size is represented by the fact, that particulated antigens taken up via phagocytosis are more efficiently presented on MHC class I molecules (Harding et al., 1994) consequently leading to more potent CTL responses than those following the loading of soluble antigens. Additionally PLGA particles potentially protect their content from extracellular degradation leading to increased efficiency of delivery (Panyam et al., 2003). Moreover it has been shown for antigens delivered by PLGA nanoparticles that they are able to escape endosomal degradation and reach the cytoplasm at significantly higher levels compared to other antigen forms leading to more efficient and significantly longer MHC-I presentation (Shen et al., 2006; Heit et al., 2007).
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Chapter 2
Encapsulation of proteins and peptides into biodegradable poly (D,L-lactide-co-
glycolide) microspheres prolongs and enhances antigen presentation by human
dendritic cells
Ying Waeckerle-Men, Edith Uetz-von Allmen, Bruno Gander, Elke Scandella, Eva Schlosser, Gunther Schmidtke, Hans P. Merkle, Marcus Groettrup
Chapter 3
Ex vivo loading of dendritic cells with peptide loaded PLGA microspheres enhances T-lymphocyte response and is not depending on maturation of dendritic
cells
Eva Schlosser, Sameh Basta, Ying Waeckerle-Men, Marcus Groettrup
1. Abstract
The use of dendritic cells externally loaded with tumour associated peptides as vaccines has been hampered by several factors. To increase antigen presentation by APCs in vivo biodegradable (PLGA) microspheres loaded with peptide were used as an antigen carrier system. In comparison to conventional vaccination regimen using peptide pulsed dendritic cells, significantly increased cytotoxic T-lymphocyte responses could be observed when PLGA loaded dendritic cells were injected.
2. Introduction
Immunotherapy using autologous dendritic cells pulsed with tumour associated peptides is hampered by a number of factors. Namely the optimal protocol for generation of clinical grade dendritic cells for reinjection as well as the ideal vaccination regimen remains still a matter of research and discussion (reviewed in Osada, Clay et al. 2006 and Tuyaerts et al., 2007). Furthermore, the migratory capacity of in vitro generated DCs from the skin into the lymph node has been shown to be rather inefficient (Morse et al., 1999; Adema et al., 2005)
Another issue is the optimal loading strategy for dendritic cells. Most clinical studies make use of external pulsing of autologous dendritic cells with synthezised MHC-I peptides. Because the half-life of MHC-I: peptide complexes is –in the order of only one day- very short, this method is associated with the disadvantage of a short duration of lymphocyte-DC interaction which might in conclusion not be sufficient for inducing an efficient immune response (Ludewig et al., 2001, Dieckmann et al., 2005). Moreover for external loading only peptides, which match to their corresponding HLA type can be used. Therefore, these therapies are restricted only to a subgroup of patients displaying the appropriate HLA and to the few epitopes known for tumour antigens. As a third point hampering the method of using external loading of MHC-I restricted epitopes, helper epitopes are missing, especially since there are very few of them known (Velders et al., 2003). Hence, it would be optimal to charge MoDCs with entire proteins that are likely to contain several MHC-class I and II epitopes and deliver the proteins in a form such that they can be taken up efficiently by DC and provide T cell epitopes over an extended period.
problems of conventional antigen delivery. The method is based on the inclusion of either synthetic peptides or proteins as antigens in PLGA-MS. PLGA is a polymeric ester consisting of the two -hydroxyacids lactic acid and glycolic acid. Hydrolysis of the polymer in aqueous solution leads to the release of the two acids which can be metabolized via the citric acid cycle (Brady et al., 1973). This material has been used for many years for the production of biodegradable surgical sutures and for the encapsulation of drugs. It is well characterized and approved for application in humans which is an obvious advantage for antigen delivery (Johansen et al., 2000).
PLGA polymers can be converted by spray drying into microspheres of a defined particle diameter of 1.5-7 µm i.e. a size that is ideal for the uptake by macrophages and DCs (Shen et al., 1997). It has been shown, that the encapsulated peptides and proteins reach the processing pathways for MHC-I and MHC-II (Men et al., 1999).
Additionally, MS have the ability to hydrolyse slowly within the cell in vivo so that they release their content for up to 2 weeks post uptake (Peyre et al., 2004), leading to a prolonged antigen presentation (Waeckerle-Men et al., 2006).
In order to test the in vivo potency of antigen delivery via PLGA-MS into DCs we prepared PLGA –MS containing either an epitope of β-galactosidase or the full length protein β-galactosidase. BMDCs derived from C57BL/6 mice were generated and pulsed with the PLGA-MS. Subsequently these cells were reinjected into animals to compare the induced immune response with the response elicited after “classical”
injection of autologous dendritic cells pulsed with peptide.
3. Results
To compare the efficiency of MS loaded DCs with the conventional peptide loading strategy, generation of bone marrow derived dendritic cells (BMDCs) from mice was established. Therefore bone marrow from C57BL/6 mice was isolated and cultured for 5 days in GM-CSF-containing medium. Phenotypic analysis of the cells showed high expression levels of MHC class I (Kb), as well as MHC class II (I-Ab) and
intermediate expression of CD80. The expression level of CD11c, an integrin serving
as a marker for dendritic cells (Metlay et al., 1990) ranged in all experiments between 70 and 90% (fig. 1).
Fig. 1. Generation of BMDCs. Representative flow cytometric analysis of BMDCs generated by culturing C57BL/6 bone marrow derived monocytes in GM-CSF containing medium. On day 5 cells were harvested and stained with monoclonal antibodies for CD11c, H2-Kb, I-Ab and CD80 and analyzed by flow cytometry.
In order to test the in vivo capacitiy of the generated BMDCs to stimulate an immune response, they were externally loaded with the I8V epitope of β-galactosidase (residues 497-504)as a model antigen and subsequently injected intravenously (i.v.) into C57BL/6 mice. Six days after injection, intracellular IFN-γ staining was used as read out system for CD8+ T-cell activation. This system is based on the fact that T- cells when activated by APCs start producing IFN-γ after a short period of restimulation in vitro. The portion of the cells producing IFN-γ is correlated with the amount of MHC-I:peptide complexes they were facing before, making it an appropriate system for measuring ex vivo immune responses. As it can be seen in fig. 2, immunisation of naïve mice with peptide pulsed dendritic cells resulted in an amount of 0,8-1,1% activated T-cells. To ensure that the observed IFN-γ production by these cells is specifically driven by the injected antigen and not due to an unspecific inflammatory response, splenocytes which were not restimulated from the same animals served as a background control (data not shown). The gates in fig. 2 were placed in such a way, that unspecific IFN-γ production was set to zero.
Fig. 2: Immunisation of mice with peptide pulsed dendritic cells elicits IFN-γ producing CD8+ lymphocytes. BMDCs (d5) were loaded with I8V-epitope and subsequently injected i.v. in C57Bl/6 mice. After 6 days splenocytes were harvested and intracellular IFN-γ production of CD8+ lymphocytes was assessed using flow cytometry. Each picture represents one BMDC injected animal.
To compare these results from the “classical” DC immunisation model (fig. 2) with the MS based approach, BMDCs were incubated with peptide loaded Microspheres overnight and used in the same vaccination protocol as described above. As shown in fig. 3 injection of MS-peptide packaged BMDCs resulted in significantly (p-value=
0,0049 after performance of students test) enhanced T-lymphocyte activation.
We also evaluated the effect of encapsulation of full length β-galactosidase into PLGA-microspheres (here referred as MS-protein) in the same experimental setting.
But surprisingly responses were with about 1% of activated T-cells above the level of immune response elicited by peptide loaded BMDCs.
Fig. 3: Injection of dendritic cells loaded with encapsulated peptide leads to significantly increased CD8+ responses compared to injection of peptide pulsed dendritic cells. BMDCs (d5) were coincubated either with encapsulated full length protein (β-galactosidase) or with the encapsulated epitope I8V (residues 497-504 of β-galactosidase) o/n. On the next day BMDCs pulsed with I8V-epitope for 2 hours and the aforementioned BMDCs were i.v. injected into C57Bl/6 animals. After 6 days, splenocytes were isolated and intracellular IFN-γ production was measured.
Waeckerle-Men et al. showed that injection of Dendritic cells pulsed with microspheres markedly enhance and prolong T-cell proliferation using stainings for the specific T-cell receptor (tetramer-stainings) (Waeckerle-Men et al., 2006).
In order to complete this study with a more functional analysis of T-cells, a kinetic was performed using two groups of animals, which either received MS pulsed or peptide pulsed DCs for 6, 8 or 15 days. Instead of tetramer staining, intracellular IFN- γ staining was chosen as the preferential read out system.
As it is shown in figure 4, on day 6 the T-cell responses of the animals which had received the MS loaded BMDCs peaked much stronger than the responses of the animals which had received the externally pulsed BMDCs.
Already on day 8 the response significantly declined in both groups, although still double the amount could be monitored in the group which was treated with MS.
Finally on day 15 only a slight response could be observed in both groups to a similar extent.
d6 d8 d15 0
1 2 3
4 DC-pep
MS-pep
% of IFN-γ producing CD8+ Lymphocytes
Fig. 4: Antigen loading of BMDCs leads to enhanced, but not prolonged CTL responses ex vivo. BMDCs were loaded externally either with encapsulated β- galactosidase or were externally loaded with peptide. After 6, 8 and 15 days of immunisation splenocytes from each mouse were collected and intracellular IFN-γ staining with subsequent flow cytometric analysis performed. Shown are the mean data from three animals.
It is known that maturation of DCs is recquired for the induction of a sufficient immune response, if absent possibly even leading to tolerance (Probst et al., 2005 ).
Additionally some groups proposed that PLGA itself may cause maturation of DCs when coincubated (Diwan et al., 2003; Yoshida et al., 2007). In order to reveal, whether the adjuvant effect of BMDCs loaded with MS, shown above, might have been due to maturation of the DCs through PLGA-MS itself, becoming manifest in upregulation of MHC-I, MHC-II as well as upregulation of costimulatory molecules like CD80, the influence of microspheres on maturation of BMDCs was assessed next.
Therefore BMDCs were either incubated with LPS as a ligand for TLR-4 or with microspheres for 18h. As it is shown in fig. 5 coincubation of dendritic cells with MS did not lead to any significant upregulation of maturation markers while incubation with LPS did. As a standard for immature BMDCs, BMDCs derived from the same animal but left untreated were used.
If one assumes that maturation by PLGA represents the crucial factor in the enhancement of immune response shown afore, loading of BMDCs with PLGA
microspheres with subsequent maturation shall not influence the immune response (because BMDCs are already matured via PLGA).
Fig. 5: Maturation of BMDCs after PLGA-MS coincubation o/n. Immature BMDCs (d5) were cocultured with empty PLGA-MS O/N (Microspheres). Next day surface expression of MHC-I (H2-Kb), MHC-II (I-Ab) and CD80 were evaluated via flow cytometry.
As negative control served immature, untreated BMDCs, as positive control LPS matured BMDCs.
To assess this hypothesis, immature dendritic cells were incubated with PLGA o/n and subsequently matured using LPS. To compare the effect, BMDCs derived from the same preparation received the same amount of microspheres but were not treated with LPS. In order to test the influence of maturation on peptide pulsed BMDCs, these were matured and subsequently pulsed with peptide followed by i.v.
injection.
As shown in fig. 6 maturation of dendritic cells after PLGA-MS pulsing did in fact lead to increased intracellular IFN-γ production compared to injection of PLGA-MS immature dendritic cells. Also the maturation of the peptide pulsed BMDCs had an enhancing effect on the immune response.
Fig. 6: Maturation of BMDCs after loading either with encapsulated or with soluble epitope I8V leads to an increased immune response. BMDCs (d5) were generated and either pulsed with epitope I8V for 1 hour (im pep) or with epitope containing PLGA-MS o/n (im MS pep). Subsequently BMDCs were matured via LPS coincubation and injected i.v. into C57BL/6 mice (ma pep and ma MS pep). After 6 days of immunisation, splenocytes were collected and intracellular IFN-γ staining was performed. Shown are the mean results from three animals +/- SEM.
4. Discussion
It has been shown that dendritic cells upon subcutaneous injection need 24-48 hours after vaccination to reach the draining lymph node in substantial numbers (Hermans et al., 2000), once they have reached the lymph node, T-cell priming needs at least 72 hours (Mempel et al., 2004; Spierings et al., 2006). Taken together robust T-cell priming needs 3 days, but external loading of autologous dendritic cells with peptides diminishes already after 48h (Kukutsch et al., 2000). Thus prolongation of Ag presentation would be of great benefit for a vaccination system.
Since it has been shown in in vitro studies that encapsulation of antigens into PLGA- MS leads to significantly prolonged and increased antigen presentation by dendritic cells and macrophages in vitro compared to external peptide loading (Audran et al.,
2003; Waeckerle-Men et al., 2006) it might be very likely to overcome the aforementioned problem with this system.
In order to investigate how loading of dendritic cells with antigen packaged PLGA-MS in vitro enables the generation of CTL responses in vivo, β-galactosidase and the synthetic H-2Kb restricted β-gal epitope I8V were encapsulated as model antigens into PLGA-MS.
The immune response after injection of PLGA-MS loaded BMDCs was compared to injection of BMDCs externally pulsed with peptide. As it is shown in fig. 3 encapsulation of peptide leads to double the amount of IFN-γ producing cells compared to loading of BMDCs with soluble peptide. This is in agreement with the study of Waeckerle-Men et al., 2006, in which injection of BMDCs loaded with encapsulated β-gal peptide also led to an increased immune response compared to injection of peptide pulsed BMDCs. Unfortunately the encapsulation of the full length β-galactosidase protein did not lead to any increase in the immune response. This was very likely due to encapsulation problems of the protein, which will be elaborated more detailed in one of the following chapters.
To investigate whether enhancement of the IFN-γ production in CD8+ lymphocytes (fig.3) by administration of antigen PLGA loaded BMDCs might have been due to the proposed immunostimulatory capacity of the PLGA itself, in vitro maturation of BMDCs by empty PLGA-MS was investigated. In contrast to other studies performed on human and mouse dendritic cells (Diwan et al., 2003; Yoshida et al., 2007), none of the maturation markers, namely MHC-I, MHC-II or CD80 was upregulated. A lack of maturation after coincubation with microspheres were also observed by others (Waeckerle-Men et al., 2004; Sun et al., 2003). Maturation of BMDCs by PLGA itself could be further ruled out by packaging the BMDCs with subsequent maturation by a ligand for TLR-4, namely Lipopolysacharide (LPS), which significantly increased the immune response, suggesting that inclusion of a maturation stimulus in the vaccination protocol would be of great benefit.
Excluding the possibility of a biomaterial adjuvant effect the increased IFN-γ response can be rather explained by enhanced delivery of antigenic material into the MHC-I presentation pathway associated with prolonged presentation of antigen (Shen et al., 2006; Waeckerle-Men et al., 2006).
positive T-cells were not detected with the IFN-γ assay.
Other studies have also revealed the induction of functional impaired T cells, which are described as a result of continued antigenic stimulation occurring during infection with adenovirus (Krebs et al., 2005; Yang et al., 2003). In terms of antigenic load and duration of antigen release MS might be comparable to chronic infection, therefore induction of non functional T cells could be likely to occur.
Nevertheless the detection of robust responses after injection of PLGA-MS pulsed dendritic cells encouraged us to continue with this strategy in a direct injection approach.
5. Materials and methods
Materials, media and mice
PLGA 50:50 of approx. 15kDa, carrying uncapped hydroxyl and carboxyl end-groups (Resomer RG502H) was purchased from Boehringer Ingelheim (Ingelheim, Germany). All media were purchased from Invitrogen Life Technologies (Karlsruhe, Germany) and contained GlutaMAX, 10% FCS, and 100U/ml penicillin/streptomycin.
LPS and β-galactosidase were bought from Sigma. H-2Kb restricted peptide I8V (β- Gal 497-504, ICPMYARV) was synthesized by Eurogentec.
C57BL/6 (H-2b) mice were originally obtained from Charles River, Germany. Mice were kept in a specific pathogen-free facility and used at 6-10 weeks of age.
Preparation of PLGA microspheres
All antigens used in this study were microencapsulated into RG502H polymer by using the spray drying technique as described elsewhere (Waeckerle-Men et al., 2004). Briefly, 5mg of antigen was dissolved in 1ml H2O. 1g of PLGA was dissolved in 20ml ethyl formate. The two solutions were mixed and homogenized under
ultrasonication (20kHz, 50 W, VibraCell VC50T, Sonics & Materials, Canbury, CT) during 2 x 30s under cooling on ice. This w/o dispersion was spray-dried using a Mini Spray-Dryer 191 (Büchi, CH-Flawil) at a flow rate of 2ml/min at a inlet and outlet temperature of 48, respectively 40 °C. The spray dried Microspheres were washed out of the spray-dryer´s Cyclone with 0,1% poloxamer 188 solution, collected on a cellulose acetate membrane filter, washed with water to remove non-encapsulated protein and dried under reduced pressure (20mbar) for 18h.
Generation of BMDCs and maturation mediated by empty microspheres
For preparation of murine CD11c+ BMDCs femurs of C57BL/6 mice were taken and bone marrow was isolated by flushing with PBS. Lysis of erythrocytes was performed using NH4Cl. Afterwards cells were cultured in 1640 RPMI medium supplemented with 10% FCS, 2-ME and 10% supernatant of GM-CSF transfected X63Ag8–653- cells. Cells were harvested after culturing for 5 days in 6 well plates at a density of 5x105 cells per ml. Percentage of CD11c+ cells was approximately 70%. For maturation, cells were cocultured with LPS (10µg/ml). To test maturation mediated by empty MS, cells were incubated with 1mg empty microspheres per ml o/n. Afterwards cells were stained with antibodies for H-2Kb (clone AF6-88.5), CD86 (clone GL1), H- 2IAb (clone AF6-120.1). All antibodies were purchased from BD Pharmingen. FACS analysis was performed with a FACScan™ using CellQuest™ software and subsequently analysed by Flowjo™ software.
Loading of BMDCs with peptide/ microspheres
BMDCs were harvested on day 5 and either loaded with Microspheres 1mg/ml ON or with I8V peptide for 2 hours at a concentration of 10-6 M. Subsequently 5x105 cells were injected i.v. into C57BL/6 mice (H-2b) i.v..
