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Departamento de Farmacia y Tecnología Farmacéutica
Facultad de Farmacia y Nutrición
UNIVERSIDAD DE NAVARRA
TESIS DOCTORAL
“Design and in vitro / in vivo evaluation of a targeted
immunotherapy platform for the treatment of melanoma”
“Diseño y evaluación in vitro / in vivo de una plataforma
dirigida de inmunoterapia para el tratamiento de melanoma”
Trabajo presentado por María Merino Díaz para obtener el Grado de Doctor
María Merino Díaz
Pamplona, 2018
UNIVERSIDAD DE NAVARRA
FACULTAD DE FARMACIA Y NUTRICIÓN
Departamento de Farmacia y Tecnología Farmacéutica
Memoria presentada por Dña. María Merino Díaz para aspirar al grado de Doctor por la
Universidad de Navarra.
Fdo. María Merino Díaz
El presente trabajo ha sido realizado bajo nuestra dirección en el Departamento de
Farmacia y Tecnología Farmacéutica de la Facultad de Farmacia y Nutrición de la
Universidad de Navarra y autorizamos su presentación ante el Tribunal que lo ha de juzgar.
VºBº Director VºBº Co-Director
María Jesús Garrido Cid Sara Zalba Oteiza
Las investigaciones realizadas en el presente trabajo se han
desarrollado dentro del proyecto “Estudio de agentes
inmunomoduladores en oncología mediante plataformas preclínicas
para desarrollar un modelo cinético-dinámico con carácter
traslacional” financiado por el Plan de Investigación de la
Universidad de Navarra (PIUNA).
A mi familia y Enrique.
AGRADECIMIENTOS
AGRADECIMIENTOS
En primer lugar me gustaría expresar mi agradecimiento a la Universidad de
Navarra y al Departamento de Farmacia y Tecnología Farmacéutica por haberme permitido
realizar esta tesis doctoral.
A la Dra. María Jesús Garrido Cid y al Dr. Ignacio Fernández Trocóniz, por
haberme dado la oportunidad de realizar la tesis en su grupo. En especial a María Jesús,
muchas gracias por la dirección de esta tesis, por guiarme y aconsejarme en estos años, y
por la confianza puesta en mí y en mi trabajo.
A los investigadores, profesores y personal del Departamento de Farmacia y
Tecnología Farmacéutica: Socorro Espuelas, Carmen Dios, Maribel Calvo, María del Mar
Goñi, Juan Manuel Irache, Elisa Garbayo, Conchita Tros, Fernando Martínez, Félix
Recarte, María Huici, Noelia Ruz y María José Blanco, por compartir día a día el lugar de
trabajo y por la ayuda prestada cuando así lo he necesitado. A Hugo Lana, gracias por tu
ayuda y por hacerme valorar el trabajo bien hecho.
A los compañeros del departamento, los que están y los que ya se han ido, Nekane,
Simón, Laura S, Carlos, Meli, Cristina, Paula, Laura I, Juana, Koldo, Leire, Itziar, Violeta,
Zinnia, Edu, Ana, JD, María G, Belén, Víctor, Nuria, Nacho, Diego, por compartir tantas
experiencias de trabajo, aprendizajes y ratos agradables a lo largo de estos años.
A todos los que han hecho posible que este trabajo saliera adelante: al departamento
de Microbiología y al departamento de Histología y Anatomía Patológica de la
Universidad de Navarra, a la Unidad de Imagen y Morfología del CIMA.
My most sincere gratitude to all the people of the Erasmus Medical Center of
Rotterdam, The Netherlands, for their hospitality and support during my stay there. In
particular to Timo L.M. ten Hagen, thank you so much for accepting me going to your lab,
giving me advice and for the confidence you placed on me. I learnt a lot working there.
Gracias a toda la gente del CIMA, Noelia, Teresa, Juanjo y Sandra, y en particular
al laboratorio 3.03, Marcos, Celia, Nuria y Kepa. Gracias por todos esos cafés y charlas y
por la ayuda y el apoyo que me habéis prestado durante todos estos años.
A las chicas del coche, Ana, Miriam y Sara G. Pero un especial cariño a Inma y a
Marina, por esas largas charlas existenciales y el apoyo, compresión y cariño que habéis
AGRADECIMIENTOS
mostrado todos estos años. ¡¡Sin vosotras los viajes diarios habrían sido interminables y os
habéis convertido en verdaderas amigas!!
A mis chihuahuas, Lina, Esther, Yolanda, Alba, Inés, Ana Luisa, Edurne L, Jorge y
Cristian, millones de gracias por los momentos que hemos vivido todos estos años. Por
esas comidas y excursiones, por apoyarme y ayudarme a intentar encontrar opciones y
salidas a tantos problemas. Pero sobre todo por vuestra amistad, por hacerme sonreír,
animarme y hacerme ver el vaso lleno cuando lo veía completamente vacío.
A Ana M y Laura B, qué diría de vosotras. Me habéis acompañado en toda esta
tesis. Millones de gracias por todo este tiempo, por lo que me habéis enseñado, vuestra
ayuda, apoyo, cariño y compresión. ¡¡Gracias por vuestra amistad!!
A Sara, qué decir de Sara Zarrrba. Me metiste en esto, y tú me has sacado…Gracias
por todos estos años, por tu paciencia, por enseñarme, por hacerme ver este mundo de la
forma que tú lo ves, por tu amistad, apoyo, ánimos y sinceridad. Sara ya sabes que sin tu
ayuda esta tesis no habría salido adelante, así que ¡¡GRACIAS POR TODO!!
A todo el AJN, en especial a Iñi, Miguel y Javi, por taaaantos y taaaantos
momentos juntos, ¡y los que nos quedan!. Pero sobretodo y con muchísimo cariño a mis
Belenchu y Alber, gracias por aguantarme incluso sin comprender lo que decía, gracias por
todos los viajes juntos, vacaciones, salidas, excursiones, cenas…¡¡Sois geniales!!
A la familia Buil-Herreros de Tejada, y muy especial a Pepa, Juan, Pauli y David.
Gracias por el apoyo que me habéis dado desde que os conocí.
Como no a mis amig@s, Sani, Ire, Car, Alex y Cris, sois y seréis los mejores
amigos que se puede tener. Gracias por haber estado ahí en todo momento, por haber
aguantado tantos y tantos audios y conversaciones sobre algo que ni entendíais, por
apoyarme en esta larga aventura y darme ánimos y esperanza. Gracias por hacerme ver el
lado positivo de las cosas y hacerme reír en momento difíciles. ¡¡¡Sois l@s mejores!!!
A mi familia, en particular a mis yayos. Habéis sido, sois y seréis una parte muy
importante de mi vida, me habéis apoyado en todo lo que he decidido hacer sin poner en
duda mi capacidad para ello, siempre alegres y contentos de poder pasar ratos juntos,
aunque ésta tesis me haya hecho perder algunos. Pase lo que pase SIEMPRE os voy a
querer.
Mis últimas palabras son para las personas más importantes de mi vida, mis padres.
Nunca os podré agradecer todo lo que habéis hecho y estáis haciendo por mí, por la
educación que me habéis dado, y los valores que me habéis sabido transmitir. Sin vosotros
AGRADECIMIENTOS
y vuestro apoyo incondicional no habría podido hacer muchas de las cosas que he hecho.
¡¡OS QUIERO!!
Y por último Enrique…qué decir de Enrique. Gracias por esas visitas a Italia,
Inglaterra y Holanda. Gracias por tu paciencia, tu amabilidad, tu comprensión y tu cariño.
Gracias por saber estar ahí en todo momento intentando sacarme una sonrisa. Gracias por
haber aguantado todos los momentos de estrés y haberme apoyado en ésta aventura ¡Nos
vemos el 29 de Junio!
A todos los que de alguna forma han formado parte de esta aventura,
¡MUCHISIMAS GRACIAS!
CONTENTS
CONTENTS
ABBREVIATIONS ............................................................................................................... 1
INTRODUCTION ................................................................................................................ 5
References ...................................................................................................................... 13
CHAPTER 1
Immunoliposomes in clinical oncology: State of the art and future
perspectives
Abstract ............................................................................................................................ 21
1. Introduction .................................................................................................................... 23
1.1. Moving from passive to active targeting ................................................................. 25
2. Methodology for immunoliposomes development ......................................................... 29
2.1. Antibody fragments for liposome coupling ......................................................................... 31
2.2. Coupling methods: Conventional and Post insertion methods ............................................. 33
2.3. Role of PEG in targeted liposomes ...................................................................................... 35
3. Mechanism of action ...................................................................................................... 38
4. Pharmacokinetics (PK) of targeted liposomes................................................................ 40
5. Clinical trials with targeted liposomes ........................................................................... 45
5.1. Transferrin-targeted liposomes ............................................................................................ 45
5.2. HER-2 and EGFR targeted liposomes ................................................................................. 47
5.3. GAH targeted liposomes ...................................................................................................... 48
5.4. EphA2 targeted liposomes ................................................................................................... 49
5.5. Glutathione targeted liposomes ............................................................................................ 50
6. Future insight for immunoliposomes .............................................................................. 50
6.1. Patients stratification for immunoliposomes administration ................................................ 50
6.2. EPR modulation to improve tumor targeting ....................................................................... 51
6.3. Stimuli-responsive liposomes .............................................................................................. 53
6.3.1. External stimuli ........................................................................................................................54
6.3.2. Internal stimuli .........................................................................................................................55
CONTENTS
6.4. Multi-targeting liposomes .................................................................................................... 56
7. Conclusion ...................................................................................................................... 57
8. Acknowledgements ........................................................................................................ 59
9. References ...................................................................................................................... 59
OBJECTIVES..................................................................................................................... 73
CHAPTER 2
A new immune-nanoplatform for promoting adaptive antitumor immune
response
Abstract ......................................................................................................................... 81
Introduction ................................................................................................................... 83
Materials ........................................................................................................................ 84
Methods ........................................................................................................................ 85
1. Preparation of ligands .................................................................................................... .85
1.1. Anti-PD-L1 mAb ................................................................................................................. 85
1.2. Anti-PD-L1 Fab’ fragments ................................................................................................ 86
2. Preparation of immunoliposomes ................................................................................. 86
3. Coupling methods .......................................................................................................... 87
3.1. Conventional method ........................................................................................................... 87
3.2. Post-insertion method .......................................................................................................... 88
4. Characterization of liposomes ........................................................................................ 89
5. Stability assay ................................................................................................................. 90
6. Affinity of ILs for PD-L1 ............................................................................................... 91
7. In vitro cell studies ......................................................................................................... 91
7.1. PD-L1 expression ................................................................................................................. 91
7.2.Cellular interaction of liposomes ......................................................................................... 92
8. In vivo studies ................................................................................................................. 92
8.1. Biodistribution study ............................................................................................................ 93
8.2. Immunological effect ........................................................................................................... 94
8.3. Therapeutic efficacy ............................................................................................................. 95
9. Statistical analysis .......................................................................................................... 95
Results ........................................................................................................................... 96
CONTENTS
1. Enzymatic digestion using pepsin provided Fab’ fragments ......................................... 96
2. Drug release was not affected by the amount of PEG ................................................... 97
3. Immunoliposomes bound selectively to PD-L1 ............................................................ 98
4. In vitro studies ............................................................................................................... 99
4.1. Baseline PD-L1 expression in inducible by IFN-γ ............................................................... 99
4.2.ILs showed PD-L1 specificity in B16OVA cells ............................................................... 100
5. In vivo studies .............................................................................................................. 102
5.1. Biodistribution patterns evaluated for ILs and non-targeted formulation .......................... 102
5.2. Fab’ ILs induced a specific immune response ................................................................... 106
5.3. Fab’ ILs were able to induce tumor shrinkage ................................................................... 107
Discussion ................................................................................................................... 109
Acknowledgements ..................................................................................................... 113
Grant and financial support ......................................................................................... 113
References ................................................................................................................... 114
Supplementary material ............................................................................................... 117
CHAPTER 3
Doxorubicin immunoliposomes against PD-L1 enhance the immune
stimulation against melanoma cancer cells
Abstract ......................................................................................................................... 131
Introduction .................................................................................................................. 133
Materials ....................................................................................................................... 134
Methods ........................................................................................................................ 135
1. Formulation of Dox liposomes ..................................................................................... 135
1.1. Anti-PD-L1 Fab’ fragments obtaining .............................................................................. 136
1.2. Doxorubicin immunoliposomes obtaining ......................................................................... 136
2. Characterization of liposomes ...................................................................................... 137
3. Dox release from liposomes ......................................................................................... 138
4. Cell line ....................................................................................................................... 138
5. In vitro studies ............................................................................................................. 139
5.1. Cytotoxicity study .............................................................................................................. 139
5.2. Cellular interaction of liposomes ....................................................................................... 139
6. In vivo studies .............................................................................................................. 140
CONTENTS
6.1. Pharmacokinetic evaluation ............................................................................................. 141
6.2. Activation of the immune system by ILs treatment ......................................................... 142
6.3. Antitumor effect ............................................................................................................... 144
7. Statistical analysis ........................................................................................................ 144
Results .......................................................................................................................... 145
1. Preparation and characterization of immunoliposomes ............................................... 145
2. In vitro studies .............................................................................................................. 145
2.1. Doxorubicin liposomes were stable in serum .................................................................. 145
2.2. LDF significantly decreased the IC50 ............................................................................... 146
2.3. PD-L1+ cells showed an increased uptake of targeted liposomes .................................... 147
3. In vivo studies ............................................................................................................... 149
3.1. PK profile depends on treatment and dose repetition ...................................................... 149
3.2. LDF achieved a systemic antitumor immune activation ................................................. 150
3.3. LDF achieved to control the tumor growth ..................................................................... 152
Discussion .............................................................................................................. 153
Acknowledgements ................................................................................................ 158
Grant and financial support .................................................................................... 158
References .............................................................................................................. 158
GENERAL DISCUSSION ............................................................................................... 163
References ................................................................................................................... 168
CONCLUSIONS/CONCLUSIONES ............................................................................. 171
ANNEX I ........................................................................................................................... 177
ANNEX II .......................................................................................................................... 195
ANNEX III ........................................................................................................................ 209
ABBREVIATIONS
1
ABBREVIATIONS
ABC Accelerated Blood Clearance
ACK Ammonium Chloride Potassium lysing buffer
ADAs Anti-drug antibodies
AUC Area under the curve plasma concentrations
CH Cholesterol
CHEMS Cholesteryl hemisuccinate
DAPI 4′,6-diamidino-2-phenylindole
DiI 1,1′-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate
DOPE Dioleoylphosphatidylethanolamine
Dox Doxorubicin
DPPC Dipalmitoylphosphatidylcholine
DPPG2 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylglycerol
DSPC 1,2-Distearoyl-sn-glycero-3-phosphocholine
DSPE-PEG2000 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-
methoxy(polyethylene glycol)-2000
EGFR Epidermal Growth Factor Receptor
EPR Enhanced permeability and retention effect
Fab´ Monovalent Antibody binding fragment
F(ab’)2 Bivalent Antibody binding fragment
FBS Fetal bovine serum
Fc´ Crystallizable fragment of an antibody
GrzB Granzyme B
HER-2 Human Epidermal Growth Factor Receptor -2
HPLC High-Performance Liquid Chromatography
ABBREVIATIONS
2
HSPC Hydrogenated Soy L-α-phosphatidylcholine
Hz-PEG Hydrazine-Polyethylene glycol
IC Immune Checkpoint
IC50 Inhibitory concentration 50
ICD Immunogenic Cell Death
IL Immunoliposome
mAb Monoclonal antibody
Mal-PEG Maleimide-DSPE-PEG2000
MEA Cysteamine hydrochloride / 2-Mercaptoethylamine HCl
MIF Mean Intensity Fluorescence
MWCO Molecular Weight Cut Off
PD Pharmacodynamics
PD-1 Programmed Death 1
PD-L1 Programmed Death Ligand 1
PDP-PEG Pyridylditiopropionoylamino-Polyethylene glycol
PEG Polyethylene glycol
PK Pharmacokinetic
RES Reticuloendothelial system
RPMI Roswell Park Memorial Institute medium
RT Room temperature
scFv Single chain antibody variable regions
SD Standard deviation
SDS Page Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEM Standard Error of the Mean
SPDP 3-(2-Pyridyldithio) propionic-acid-N-hydroxysuccinimide-ester
ABBREVIATIONS
3
SRB Sulforhodamine B
TBS Tris Buffer Saline
TCEP Tris (2-carboxyethyl) phosphine
5-FU 5 -Fluorouracil
INTRODUCTION
INTRODUCTION
7
INTRODUCTION
Cancer is one of the principal causes of death in developed countries 1. Most of the
anticancer agents used nowadays exert interesting antitumor activity, associated with
serious side effects that limit, in some cases, the use of these therapies 2,3
. Chemotherapy is
characterized by having a large volume of distribution that leads to the dissemination and
death of both, healthy and malignant cells, without discrimination across them. The
concept of fighting cancer has changed over the years in order to reduce toxicity,
improving the life quality of the patients treated with chemotherapy. In this way, the
encapsulation of these molecules in different types of nanocarriers, in particular liposomes,
has provided more specific and safe treatments, some of which have achieved clinical
application 4,5
. Liposomal formulations are capable of increasing drug efficiency while
reducing toxicity 6. These nanocarriers, often composed of cholesterol (CH) and
phospholipids, are biocompatible and low immunogenic vehicles that can carry or
transport, by encapsulation, a large number of molecules with different physicochemical
properties 4,6
. The main advantage of liposomes is their capability to be accumulated in the
tumor area, enhancing the therapeutic drug activity. This is a consequence of changes in
the pharmacokinetics (PK) properties of the encapsulated agent, in particular a reduction in
the distribution. In addition, the modification of current liposomes by the inclusion of
certain polymers, such as polyethyleneglycol (PEG) covering their surface, provides an
increment in their circulation half-life in comparison to early liposomes, which were
rapidly recognized and removed by the reticuloendothelial system (RES) 4,7,8
.
Pegylated liposomes have some favourable characteristics for cancer therapy, such as low
toxicity, improved PK, long-term circulation in blood and tumor accumulation, this last
one promoted by the Enhanced Permeability and Retention effect (EPR) (Figure 1) 4,6,9
.
The EPR effect is based on the presence of a high permeability on the vascular
INTRODUCTION
8
endothelium in tumor due to the rapid growth of vessels, which results in the formation of
fenestrated and leaky blood vessels. This characteristic enables the extravasation of
molecules with a size up to 300 nm to the tumor area, which are accumulated there due to
the lack of lymphatics 10–12
.
Figure 1. Schematic representation of the EPR effect: A) Healthy tissue; B) Tumor tissue.
However, despite the tumor accumulation, generally, liposomal formulations present high
stability, limiting the intracellular drug bioavailability, and thereby the antitumor effect is
suboptimal. This fact has led to modify these liposomes by the inclusion of different types
of lipids in their composition, which allow the control of the drug release. Thus, when
certain stimulus is applied (ex. temperature, light) or is present in the tumor (pH change,
enzymes), these lipids change their conformation and destabilize the liposome membrane,
driving to the rapid release of the cargo 4,13–16
. However, despite these advantages of
improving the rapid release at the tumor site, the lack of selectivity and specificity of these
INTRODUCTION
9
stimulus-sensitive formulations may lead to toxicities in tumor surrounding healthy tissues
by the non-internalized drug, which is cleared by systemic circulation 6,17
.
In this regard, liposomes can be functionalized with different molecules (antibodies,
peptides, proteins or carbohydrates) attached to their surface to obtain targeted liposomes,
triggering a specific tumor cell recognition and interaction. However, to achieve a
therapeutic benefit, the target of the ligand coupled to liposomes must be overexpressed,
upregulated or be exclusive of tumor cells in response to a high metabolic demand or
mutations 4. Thus, liposomes targeted with certain proteins, aptamers or carbohydrates
allow tumor-specific delivery to the target sites, bypassing normal healthy cells 6,9
.
Figure 2. Schematic representation of the different fragments of a monoclonal antibody: A) Whole
mAb; B) F(ab’)2 fragment; C) Fab’ fragment; D) ScFv fragment.
This procedure triggers an increment on the therapeutic index of the encapsulated agent
6,17, being accumulated at the tumor site (based on the EPR effect) and facilitating the
specific uptake of targeted liposomes by malignant cells 6. Among the different ligands,
monoclonal antibodies (mAbs) and their monovalent variable fragments are the most
widely targeting moieties (Figure 2) used to develop targeted liposomes, known as
immunoliposomes (ILs) (Figure 3) 4,6,9,17
.
INTRODUCTION
10
Figure 3. Evolution of liposomes: A) Plain liposome; B) Pegylated long-circulating liposome; C) ILs.
Although ILs showed to be more advantageous formulation than non-targeted liposomes,
few have been commercialized. Currently, those conjugated with HER-2, GAH and
Epidermal Growth Factor (EGF) are involved in several clinical trials with promising
results 4,17
.
In this strategy, the liposome acts as a drug carrier and the ligand, such as the antibody, as
the driver to anchor the formulation to the target 17
. Nevertheless, this work proposes a step
further in ILs antitumor fight: the use of an antibody moiety not only to target but also to
exert a specific effect. In that sense, Immunotherapy is emerging as a promising approach
against many types of cancer 1, relying on the capacity of the immune system to eliminate
cancer cells 18
(Figure 5).
The immune system is able to identify specific tumor antigens from highly immunogenic
cancer cells, and kill them at an incipient status by the immunosurveillance process, which
shapes the cycle of immunity described by Chen & Mellman (2013) 19–21
. However, tumors
composed of weakly immunogenic malignant cells are characterized by a high frequency
of genetic and epigenetic abnormalities, which lead to the establishment of a process
referred to as “cancer immunoediting” 19,21
, which enables tumor escape from the immune
system. In this case, tumors provide mechanisms to transform effector T cells in non-
effective cells either by inactivation or by hyper-activation, leading to their dysfunction. In
this scenario, several factors in the tumor microenvironment can negatively modulate the
activated antitumor T cells, contributing to the immune escape of malignant cells 22
.
INTRODUCTION
11
Immune checkpoint (IC) molecules such as Programmed Death-1 (PD-1) and its ligand,
Programmed Death-Ligand 1 (PD-L1) 23
are involved in the crosstalk of immune cells and
tumor. PD-L1 is a molecule commonly overexpressed in tumor cells that binds to PD-1, a
receptor present in activated T cells. PD-L1/PD-1 binding downregulates T cell activity,
leading to T cell exhaustion or dysfunction 18,24,25
. According to this feature, several
monoclonal antibodies have been approved to block these ICs and can be conjugated into
ILs in order to control the immunosuppressive mechanisms 19,26
(Figure 4).
Figure 4. Representation of the interaction mechanism between PD-1/PD-L1: A) Downregulation of T
cell activity; B) Reactivation of the immune system.
Thus, Pembrolizumab and Nivolumab, which recognize and bind PD-1, together with
Atezolizumab, an anti-PD-L1 mAb recently approved by the FDA for the treatment of
mesothelial tumors, have demonstrated impressive clinical outcomes 23,25,27–29
. However,
INTRODUCTION
12
their use in monotherapy has led to a low amount of patients who experience therapeutic
benefits, supporting the necessity of introducing combinatorial approaches 23,30
.
In this way, some chemotherapeutic drugs, like Doxorubicin (Dox), have the ability to
induce immunogenic cell death (ICD) producing apoptosis and inflammation 31,32
, which
contributes to stimulate the immune system against tumor cell-death antigens, leading to
the activation of T cells 32–34
(Figure 5). Thus, the combination of ICD compounds with
immunotherapy, like IC inhibitors, is coined Chemoimmunotherapy. This is a new
emerging rational therapeutic approach 35
that has not been assayed in depth yet. However,
this association may provide a comprehensive understanding of the underlying antitumor
mechanisms.
Figure 5. Schematic representation of the immune response after chemoimmunotherapy treatment.
(CRT: Calreticulin; HMGB1: High-mobility group box 1 protein; ATP: Adenosine triphosphate; ICD:
Immunogenic Cell Death; IFN-γ: Interferon-gamma; IL-2: Interleukin-2; TNF: Tumor Necrosis
Factor)
According to this, the proposal of this work has been the combination of immunotherapy
and chemotherapy, applying the advantages of nanotechnology. Thus, we hypothesize that
liposomes encapsulating Dox and coupled to anti-PD-L1 at their surface would induce
tumor regression by a dual mechanism, specifically killing malignant cells and stimulating
INTRODUCTION
13
the immune system by the blockage of PD-L1, that would reduce the immunosuppressive
environment 9. In our knowledge, this is a new concept for ILs that represents the main
contribution of this project.
To achieve the present aim, several steps represented in the different chapters have been
addressed. Firstly, a detailed review about the state of the art of ILs, as well as a summary
about the main key points that have to be controlled to develop adequate ILs, is reported in
Chapter 1.
Chapter 2 is focused on the development of different immunoliposomes coupled with the
anti-PD-L1 mAb and its Fab’ fragment, using different coupling methods and PEG
percentages, to be later assayed in in vitro / in vivo studies.
Here, factors such as drug encapsulation and ligand density have been optimized, making
an important effort to achieve a formulation able to deal with adequate characteristics to
reach the tumor area and produce efficient intracellular drug delivery and immune activity.
Finally, in Chapter 3, the formulation selected in the previous work has been combined
with Dox as a proof of concept, to evaluate its in vitro / in vivo activity. This immune-
nanoplatform encapsulating Dox and targeted against PD-L1 showed a dual mechanism
characterized by an immune stimulation and a cytotoxic activity, both contributing to
reduce and eliminate cancer cells.
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INTRODUCTION
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CHAPTER 1
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE
OF THE ART AND FUTURE PERSPECTIVES
CHAPTER 1
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART
AND FUTURE PERSPECTIVES
María Merino1, Sara Zalba
1, María J. Garrido
1*
1Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy and Nutrition,
University of Navarra, Pamplona, Spain
Published in Journal of Controlled Release
Keywords: Liposome, Monoclonal Antibody, pharmacokinetic characteristics, Clinical
applications.
*Corresponding author: Address: Department of Pharmacy and Pharmaceutical Technology,
University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain. Tel.: +34 948425600: 6529. E-mail
address: mgarrido@unav.es (María J. Garrido)
CHAPTER 1
21
ABSTRACT
Liposomal formulations entrapping a vast number of molecules have improved cancer
therapies overcoming certain pharmacokinetic (PK) and pharmacodynamic limitations,
many of which are associated with tumor characteristics. In this context,
immunoliposomes represent a new strategy that has been widely investigated in
preclinical cancer models with promising results, although few have reached the stage
of clinical trials. This contrasts with the emerging clinical application of monoclonal
antibodies (mAbs). This formulation allows the conjugation of different mAbs or
antibody derivatives, such as monovalent variable fragments Fab’, to the polymers
covering the surface of liposomes. The combination of this targeting strategy together
with drug encapsulation in a single formulation may contribute to enhance the efficacy
of these associated agents, reducing their toxicities.
In this paper, we will consider how factors such as particle size, lipid composition, and
charge, lipid-polymer conjugation, method of production and type of ligand for
liposome coupling influence the efficacy of these formulations. Furthermore, the high
inter-individual variability in the tumor microenvironment, as well as the poor
experimental designs for the PK characterization of liposomes, makes the establishment
of the relationship between plasma or tumor concentrations and efficacy difficult. Thus,
adequate dosing regimens and patient stratification regarding the target expression may
contribute to enhance the possibility of incorporating these immunoliposomes into the
therapeutic arsenal for cancer treatments. All these issues will be briefly dealt with here,
together with a section showing the state of the art of those targeted liposomes that are
coming up for testing in clinical trials. Finally, some insights into future developments
such as the combination of specificity and controlled release, based on the application of
different stimuli, for the manipulation of stability and cargo release, will be offered.
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
22
This has been included in order to highlight the new opportunities for targeted
liposomes, including immunoliposomes.
CHAPTER 1
23
1. INTRODUCTION
Over the years, chemotherapy based on conventional anticancer drugs has demonstrated
good clinical efficacy. However, these molecules present serious side effects associated
with their wide and non-specific body distribution, leading in general, to a dose-
limitation or discontinuation of treatment. Thus, in order to reduce chemotherapeutic
toxicity, some of these drugs have been encapsulated in nanoparticles such as liposomes
1.
Liposomes are biocompatible and lowly immunogenic hollow vesicles usually made up
of phospholipids and cholesterol (CH). They are able to encapsulate a wide range of
drugs 2,3
that provide important advantages, in particular, those related to
pharmacokinetics, such as the reduction of the volume of distribution and the extension
of blood circulation time. All of these contribute to the enhancement of the therapeutic
index, providing a higher accumulation in the tumor and protecting healthy tissues from
side effects 1,2
.
However, conventional liposomes, rapidly recognized by opsonins and removed by the
reticuloendothelial system (RES), were modified by the addition of gangliosides such as
GM1 4 or polymers as is the case of polyethylene glycol (PEG). This change entails the
presence of a hydrophilic layer surrounding the liposome, delaying opsonization and,
thereby, RES removal (see Figure 1). This leads to an increase of the circulation half-
life of the formulation from hours to days, forming the so-called long-circulating
liposomes 5,6
.
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
24
Figure 1. Schematic representation of the opsonization process. Opsonized liposomes are able to
adhere to phagocytes through opsonin receptors, as mannose and scavenger receptors 7, and are
removed by the RES in the case of: A) conventional or non-pegylated liposomes; B) pegylated
liposomes, which are described in particular detail in the schema represented in panel C.
Since these modified liposomes have demonstrated to equal or even improve the
antitumor effect compared to the free drug in preclinical and clinical studies 2, several
liposomal formulations have been approved for the treatment of different diseases.
Table 1 lists some of the current liposomal formulations in clinical use or involved in
clinical trials.
CHAPTER 1
25
Table 1. The most popular approved liposomal formulations in clinical oncology.
Active drug Product name Indication
Annamycin Annamycin liposomes Doxorubicin-resistant tumors
Cytarabine Depocyt Cancer therapy
Daunorubicin
Daunoxome
AIDS-related Kaposi’s sarcoma, breast and lung cancer
Daunorubicin/ Cytarabine Vyxeos Acute myeloid leukemia
Doxorubicin (non-PEG liposomes)
Myocet
Combinatorial therapy for recurrent breast cancer
Doxorubicin (PEG- liposomes) Doxil/ Caelyx
Refractory Kaposi’s sarcoma, refractory ovarian cancer, recurrent breast cancer,
multiple myeloma
Doxorubicin Evacet Metastatic breast cancer
Irinotecan Onivyde® Metastatic pancreatic cancer
Lurtotecan NX211 Ovarian cancer
Plasmid encoding HLA-B7 and β2 microglobulin
Allovectin-7 * Metastatic melanoma
Platinum derivative (NDDP) 8 Platar Solid tumors
Tretinoin AtragenTM
Kaposi’s sarcoma , acute Pro-myelocytic leukemia, non-
Hodgkin’s lymphoma, renal cell carcinoma
Vincristine Marqibo® Acute lymphoblastic leukemia
Vincristine Onco TCS Non-Hodgkin’s lymphoma
Vincristine VincaXome Solid tumors
* Allovectin-7 is an immunotherapeutic agent; NDDP: cis-bis-neodecanoato (trans- R, R-1, 2-diaminocyclohexane)-
platinum II.
1.1. Moving from passive to active targeting
Cancer cells, characterized by rapid replication, have a high demand for nutrients and
oxygen for tumor growth. In this process, there is acidification and hypoxia in the tumor
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
26
microenvironment. These stimuli promote an increase in tumor vascularization, known
as angiogenesis or neovascularization. However, this neovascularization results in
immature tumor vessels that differ from healthy vessels 9,10
with regard to their
permeability and organization. Tumor vessels lack the tight junctions between
endothelial cells, giving rise to fenestrated and leaky blood vessels, irregular in shape,
with no smooth-muscle layer 9–11
. These particular characteristics allow the
extravasation of particles with a size up to 300 nm from blood to the tumor area. This,
together with the absence of proper lymphatic drainage, leads to a higher accumulation
of long-circulating nanoparticles in the tumor. This represents the basis of the Enhanced
Permeability and Retention (EPR) effect, responsible for the Passive accumulation of
liposomes and other nanoparticles in tumor tissue 9,10,12
(more detailed info in Section
6.2).
However, there is not always a correlation between the presence of nanoparticles in the
tumor microenvironment and an increase in intracellular drug bioavailability 13,14
. This
may be due to two different situations. The first can be related to the high stability of
liposomes, which leads to poor and slow drug release; whereas the second might be
associated with the low cellular uptake efficiency of liposomes. To overcome this last
obstacle, “Active Targeting” o ligand-based targeting 3,14–16
has emerged as one of the
most promising strategies to improve specific drug internalization 17
. In this approach,
targeted liposomes are able to directly bind to cancer cells. For this, the surface of the
liposomal formulations has to be decorated with different types of ligands, which
specifically recognize and bind those molecules expressed or over-expressed on the
surface of cancer cells 18
. Thus, some of these molecules, such as growth factor
receptors, are upregulated in response to an increased metabolic demand and are
associated with a poor prognosis in cancer. In addition, there are also certain epitopes,
CHAPTER 1
27
antigens, or receptors on the membrane of tumor cells which represent attractive targets
for the functionalization of liposomes 4,11
. Epidermal Growth Factor Receptor (EGFR),
Folate Receptor (FR) or Transferrin Receptors (TfR), among others, represent the most
commonly used receptors for liposome targeting. In this way, Epidermal Growth Factor
(EGF), Folate and Transferrin are the corresponding ligands for decorating these
liposomes 7.
However, due to the high mutational rate, cancer cells also express exclusive membrane
markers suitable for targeting, which improve the specificity of these targeted
liposomes. As a result, the establishment of a parallel process involving the
identification of these molecules for developing specific agents or ligands to block or
inhibit their activities is of particular interest 19
. Thus, the impact of DNA-techniques
for engineering more specific molecules has been particularly marked in the
development of monoclonal antibodies (mAbs) 20
. At that point, the coupling of mAbs
to the surface of liposomes represents the basis of “Immunoliposomes” 21
, a particular
type of targeted liposomes.
It is worth pointing out that the relevance of mAbs in cancer treatment is increasing
exponentially, despite their side effects, in particular, immunogenic reactions 22,23
.
Combinatorial therapies including mAbs and other agents, such as cytotoxic molecules,
are very often applied in patients. This is why immunoliposomes may represent an
interesting strategy, although currently, they are still in preclinical or initial clinical
phases. Table 2 lists the most common mAbs or antibody fragments conjugated to
different doxorubicin liposomes, reported in the literature 11
.
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
28
Table 2. Summary of the main immunoliposomes reported in the literature for preclinical studies.
Targeting receptors are alphabetically ordered.
Receptor Ligand Drug Type of cancer Tumor
accumulation * Coupling efficiency
Ref.
CD19
mAb Doxorubicin B-lineage
malignancies (B-cell leukemias / lymphomas and
Multiple Myeloma)
N.A. 110 µg mAb/µmol
PL 24
mAb Fab’
Doxorubicin / Vincristine
3 50-70 % Fab’
80-90 % mAb
25
mAb Doxorubucin /
Vincristine N.A.
60 µg mAb/µmol PL
26
CD20 mAb Doxorubicin B-cell Lymphoma ND 54 µg mAb/µmol
PL 24,26
CD74 Fab’ Doxorubicin B-lymphoma 6 (24h later) 60 Fab’/LP 27
EGFR Fab’ Doxorubicin
Epithelial tumors 6 (24h later) 30 µg Fab’/µmol
LP 28
GAH F(ab’)2 Doxorubicin Advanced gastric
cancer N.A.
0.02 µg F(ab)2/µg lipids
29
GD2
mAb Fab’
Doxorubicin
Neuroblastoma 10
0.33-0.53 nmol mAb/μmol PL
0.54-0.91 nmol Fab’/µmolPL
30,31
mAb c-myb Antisense
ODN 60-80 µg
mAb/µmol PL 32
HER-2 Fab’ scFV
Doxorubicin Breast cancer N.A. 30-50 Fab’/IL
20 scFv/IL 33,34
VEGFR Fab’ Doxorubicin Tumor-associated endothelial cells
10 300 Fab’/LP 35,36
2C5 (ANAs)
mAb Doxorubicin Cancer cell
specific nucleosomes
2 (24h later) 80-100 mAb/LP
37,38
5D4 mAb Doxorubicin Prostate cancer N.A. 100 mAb/LP 39
PL: phospholipid; IL: immunoliposome; LP: liposome; EGFR: Epidermal Growth Factor Receptor; GAH:
Genetically Altered Hybridoma; GD2: Disialoganglioside 2; HER2: Human Epidermal Growth Factor Receptor 2;
VEGFR: Vascular Endothelial Growth Factor Receptor; ANAs: Antinuclear Autoantibodies; Fab’: Antigen Binding
Fragment; scFv: single chain Fv; ODN: Oligodeoxynucleotides; N.A.: non-available.
* fold higher compared with non-targeted liposomes.
CHAPTER 1
29
Therefore, targeted liposomes have shifted the concept of specificity and selectivity for
tumor drug delivery, providing higher tumor recognition and subsequent cell uptake
compared to non-targeted formulations, that may lead to high drug tumor accumulation, as
is reported in Table 2 18.
2. METHODOLOGY FOR IMMUNOLIPOSOMES DEVELOPMENT
Over recent decades, the methods developed for targeted liposome formulation, including
immunoliposomes, have been changing in order to achieve higher coupling efficiency for
the different ligands, more stable formulations and methods which may be easily scalable.
Initially, ligands were directly attached to the headgroups of lipids for conventional
liposomes. However, the pegylation process changed the concept of ligand coupling.
Although one of the limitations was low ligand availability due to the ligand being hidden
in the polymeric cover, currently ligand molecules are exposed to the external surface of
targeted liposomes, avoiding sterical hindrance and guaranteeing recognition of the target
1,11,19,25,26. For this reason, ligands are currently attached to the end of PEG chains to
minimize a possible poor binding capacity to the target. Moreover, the combination of
PEG with different molecular weights has also been reported by some authors to obtain
immunoliposomes with a higher targeting efficiency 40–42
. Thus, the use of one PEG
derivative that is longer than the other, with only one being functionalized has been
implemented.
Therefore, the main strategy that has been adopted is the use of an end-functionalized
pegylated lipid. This lipid derivative is able to form a covalent bond with the ligands,
which normally requires previous activation or modification 23,24
. In general, targeted
liposomes are formulated with 1% of end-functionalized pegylated lipid. However, the
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
30
length and amount of PEG play a significant role regarding ligand-target binding ability.
This issue needs further comment and will be dealt with fully in section 2.3.
Table 3. Brief summary of the main linkers used for mAb ligand coupling during immunoliposomes
development.
PEG-derivatives Strategy Coupling efficiency
Ligand Ref.
Hz-DSPE-PEG Oxidation mAb 22-30 µg mAb/µmol PL N.A. 47
PDP-DSPE-PEG Reduction of PDP groups
conjugated with mAb-Mal micelles
93-96 µg mAb/µmol PL N.A. 47
Mal-DSPE-PEG Thiolation mAb 5-25 µg protein/µmol
lipid Cetuximab
or EGF 48,49
Mal-PEG-CH Thiolation mAb 25-35 µg mAb/µmol
lipid Cetuximab
45
Cyanur-DSPE-PEG PEG nucleophilic substitution 30-35 µg protein/µmol
lipid
Anti-E-selectin
mAb
50
Folate-PEG-CHEMS Reaction of
FBP-mAb with folate-LP 25 µg FBP-C225/µmol
lipid Cetuximab
(C225) 51
PL: phospholipid; FBP: folate binding protein; EGF: epidermal growth factor; LP: liposomes; N.A.: non-available.
In line with this, ligands such as mAbs have been coupled to immunoliposomes using
different PEG derivative linkers, as shown in Table 3. For instance, Hydrazide (Hz)-PEG,
pyridylditiopropionoylamino (PDP)-PEG, maleimide (Mal)-PEG and cyanur-PEG are the
most common lipids associated with PEG reported in literature. Additionally, folate-PEG-
CHEMS 43
, Cholesterol-anchored PEG 44
, or even the maleimide-PEG-cholesterol (mal-
PEG-CH) are also used, although they are less relevant 45
. Moreover, in recent years, it has
been reported that CH anchors are less stable when they reach the blood stream. This is
due to their hydrophobicity, which may modify the solubility of the ligands anchored to
CH, affecting their biodistribution, pharmacokinetics and efficacy. Therefore, the delivery
CHAPTER 1
31
mechanism of these CH-anchored conjugates may alter their interaction with proteins,
cells, receptors and membranes 46
.
Depending on the linker, covalent and non-covalent binding can be achieved. Thus, PDP-
PEG, Mal-PEG and Hz-PEG are the most common PEG derivatives for covalent
conjugation, whereas, Cyanur-DSPE-PEG, Folate-PEG-CHEMS and Biotine-PEG are
those most frequently used for non-covalent coupling (Table 3).
Note that for immunoliposomes, the coupling efficiency may be affected by the particular
method applied for mAb activation, because depending on the location of these activated
groups, this process can also influence targeting efficiency. Thus, although these activated
groups are arbitrarily distributed throughout the whole ligand structure, sometimes a
random orientation is given during liposome conjugation 4,27
, hindering receptor
attachment and hence, reducing specificity and targeting efficacy. However, to overcome
the random orientation of the mAb for the coupling, monovalent variable fragment of
mAbs, Fab’, can be used.
On the other hand, there are methodologies providing ligand conjugation without any
previous modification. These can achieve a higher ligand-receptor binding efficiency, as
occurs with the cyanuric chloride-DSPE-PEG linker 50
.
2.1. Antibody fragments for liposome coupling
Despite the limitations for the conjugation of the whole mAb to immunoliposomes, this
strategy is still a very promising therapeutic approach 48
, even when the presence of both
the Fc fragment and the required activation of the mAb may decrease residence time in
blood and formulation specificity, respectively (see in section 4). In order to improve these
characteristics, Fab’ fragments seemed to be one of the main solutions.
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
32
Figure 2 shows the process involved in obtaining Fab’. This starts with the enzymatic
digestion of the mAb to collect the F(ab’)2 fragments, which are then reduced using agents
such as β-mercaptoethanol or SDS, resulting in the monovalent Fab’ fragment. In this last
step of the process, F(ab’)2 fragments are also treated with β-mercaptoethylamine
hydrochloride (MEA), to specifically reduce the sulfhydryl groups between the light chains
of these bivalent fragments, allowing the exposure of -SH radicals for a correct coupling
process, and keeping receptor recognition available 24,28
.
In our experience, mAb fragmentation using pepsin may be associated with some technical
problems. This enzyme is difficult to remove during fragment collection and interferes in
protein quantification and fragment identification in western-blot analyses. At this point,
the use of immobilized pepsin is highly recommended, ensuring the absence of this protein
in the samples collected.
(MEA: 2-Mercaptoethylamine•HCl).
Figure 2. Schematic representation of two enzymatic digestion processes for obtaining different mAb
fragments: (A) pepsin and (B) papain.
CHAPTER 1
33
Smaller mAb fragments such scFv, Single domain antibodies (sdAbs), nanobodies or a
Phage Display Library have also been used for this targeting approach 52–54
. The lower
molecular weight of these ligands allows the number of molecules conjugated per liposome
to be increased in order to compensate for the monovalency of these molecules 55,56
.
Nevertheless, it has been widely reported that only 10-20 molecules of ligand per liposome
seem sufficient to achieve an efficient targeting effect 55
.
2.2. Coupling methods: Conventional and Post-insertion methods
Immunoliposomes can be formulated following the two main methods reported in the
literature, as is shown in figure 3:
Conventional method: This includes a DSPE-PEG derivative in the lipid composition
of liposomes, providing that approximately 50% of this end-functionalized PEG is
orientated to the inner space of the formulation; ligands such as mAb or Fab’ need to
be activated for their attachment to the previously formulated liposomes.
Post-insertion method: This consists of two steps: first, an end-functionalized DSPE-
PEG derivative forms micelles, which are coupled to the ligand 19,30
; second, targeted
micelles are incorporated into previously developed liposomes by a single incubation
29.
In general, the main advantage of the post-insertion strategy is the flexibility for the ligand
to be coupled to any type of liposomes, encapsulating different therapeutic agents in a
single step.
However, for both methods, the stability of the end-derivative lipid must be considered
during these coupling procedures. Thus, although 50-63% of active maleimide groups
remain on the surface for the conventional method, their activity decreases to 32% during
the purification and coupling processes. In contrast, for the post-insertion method, this
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
34
effect is minimized, with 76% of maleimide groups showing activity for the coupling
reaction 57
. However, a small amount (2-5%) of the encapsulated drug during these
processes might be lost 58
.
Figure 3. Schematic representation of the two main methods described for the development of
immunoliposomes coupled to mAb or Fab’-fragment. A) Conventional method; B) Post-insertion
method.
CHAPTER 1
35
2.3. Role of PEG in targeted liposomes
At the early stages of liposome development, the erythrocyte glycocalyx was mimicked to
obtain stealth liposomes by using polysaccharides, gangliosides and hydrated
phosphatidylinositol 59,60
. However, in order to obtain a feasible procedure for large-scale
manufacture, several polymers were tested to prolong the circulation time of nanoparticles
in blood 61
. For this reason, the PEG polymer was selected and, currently, it is the most
widely used in pharmaceutical applications due to its biocompatibility and stealth
properties. The molar mass of PEG is variable depending on the type of conjugation, 20-50
kDa for small molecules, and 1-5 kDa for liposomes or nanoparticles 61
. Although there are
several studies evaluating the impact of the different lengths of PEG on liposomal
formulations 62
, PEG-2000 is the most commonly used 59,63
.
The amount of polymer has also been reported as another factor influencing certain
characteristics of the liposomal formulations, because it can adopt two different spatial
conformations. For PEG concentrations below 5-8%, the polymer has a mushroom-like
conformation, whereas for higher concentrations, the most favorable conformation is
brush-like, as is shown in Figure 4 6,64–66
. The PEG in a mushroom structure exhibits a
globular shape, overlapping with others, and hence, covering the nanoparticle surface 59
. In
contrast, the brush conformation, found for higher amounts, is provided by interactions
between PEG chains, which show an elongated shape. Thus, in the case of percentages
higher than 10%, PEG chains present lateral repulsion destabilizing the lipid bilayer 59
.
Moreover, it has been reported that the addition of higher PEG concentrations to the lipid
mixture may promote a change in liposome structure. Thus, PEG concentrations around
15% lead to a mixture of two different size populations 67
, which correspond to liposomes
together with micelles, also called “discoidal micelles” 68
. Further, for PEG concentrations
higher than 20%, liposomes disappear, significantly decreasing particle size and inducing
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
36
the formation of spherical micelles 68
. Thus, the standard condition used for liposomal
formulations is 5% of PEG although it has been reported that 2% is sufficient to achieve a
prolonged circulation. However, this factor should be optimized for each formulation,
testing a range between 2-8% of polymer.
The PEG structure can be also modified by the presence of the corona effect. This effect is
due to the proteins that can attach to the PEG, extending or collapsing its structure and
forming a shell as a corona, a process which is both very dynamic and complex 64–66
. This
phenomenon takes place very rapidly and can mediate the interactions between liposomes
and their environment, strongly influencing the association with cell surface or even
intracellular uptake 63,69
. Therefore, a particular role is played by the PEG in
immunoliposomes and targeted liposomes, in general, because their binding capability may
be highly influenced by the type and amount of this polymer, as well as the corona effect 70
and the nature of the ligand 50
. Note that small ligand molecules can be embedded into the
PEG layer 60,71
or can display steric hindrances during the coupling step, shielding the
recognition between the DSPE-PEG derivative ending and the ligand.
However, despite its advantage for extending the circulation half-life and ligand coupling
6,64,66, PEG may reduce cell interaction, endosomal escape, and drug release, leading to a
decrease in the therapeutic drug index. This different behaviour at the pharmacokinetic and
pharmacodynamic levels reflects the “PEG dilemma”, a contradictory effect associated
with this polymer 72
. To overcome this dilemma, the cleavable PEG molecules have been
developed as a strategy able to release the polymer into the tumor tissue or even into the
endosome. Most of these cleavable PEG derivatives are designed to respond to certain
extracellular or intracellular microenvironmental conditions, such as acid pH, enzymatic
activity, and others 72
. Once the PEG is eliminated from the liposomes, they are able to
CHAPTER 1
37
interact with the tumor cell or the endosome. This strategy allows the enhancement of the
antitumor activity of pegylated liposomes 72–74
.
Figure 4. Schematic representation of the PEG conformation. A) Brush conformation; B) Mushroom
conformation.
Nevertheless, clinical studies with pegylated nanoparticles have shown an unexpected
immunogenic reaction, referred to as the Accelerated Blood Clearance (ABC)
phenomenon, which is associated with repeated administrations 75
. The mechanism is
triggered by the first administration of pegylated liposomes. These accumulate in the
spleen inducing the development of M immunoglobulins (IgM). With the second dose,
these IgM may produce immune complexes, which are rapidly cleared, reducing the
efficacy of the treatment 75,76
. Currently, to reduce the impact of this ABC effect, several
studies suggest that the dosing time for the second dose must be before the day 4 or later
than the day 7 after the first dose 75
. Interestingly, this effect does not occur for the third
dose or for certain formulations encapsulating doxorubicin, where the ABC is decreased
75.
In sum, the development of immunoliposomes represents a complex process, where factors
such as lipid composition, PEG amount, ligand coupling method, type of ligands and even
encapsulated drug must be considered in order to obtain the most suitable formulation for
attaining a specific desired effect 6,62,64
.
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
38
3. MECHANISM OF ACTION
Target recognition in tumor cells by the ligands coupled to immunoliposomes results in the
formation of a target-ligand complex. In general, this complex is internalized by
endocytosis after multi-binding stimulation. This process is highly dependent on the nature
of the ligand-receptor interaction 77,78
, as well as the path followed by the formed
endosome. This means that receptor-mediated internalization can either result in a clathrin-
dependent endocytosis, as occurs for nanoparticles modified with Tf, leading to a lysosome
formation, degrading, translocating or destabilizing the complex, that releases the drug into
the cytosol and exerts both therapeutic and toxic effects 78
, as is shown in Figure 5; or in
caveolae-mediated endocytosis transport. This latter path allows immunoliposome
internalization, bypassing lysosomes and increasing intracellular drug delivery.
Thus, some liposomal formulations include endosomal escape lipids such as N-glutaryl-
phosphatidylethanolamine (NGPE), DOPE/CHEMS or Listeriolysin O, that are able to
disrupt the lysosome, releasing the cargo into the cytoplasm 79
. Moreover, once the
complex is destabilized, receptors can be recycled to the cell surface or degraded. Note that
for certain growth factor receptors, their down-regulation involves in some cases an
antitumor effect itself.
For targeted liposomes, it is widely assumed that complex internalization must occur in
order to achieve a higher anticancer effect than in the non-targeted approach. However,
experimental evidence suggests that cellular internalization might not always be necessary,
with cellular binding or interaction being sufficient to increase drug efficacy 55,56
. Thus, in
the case of liposomes targeted with non-internalizing ligands, after specific cell binding,
their contents can be released in close proximity to other cells into the tumor
microenvironment, with subsequent drug internalization, leading to cell death and localized
damage 80
.
CHAPTER 1
39
Figure 5. Schematic representation of the mechanism triggered by the complexes formed by the ligand-
receptor interaction between targeted liposomes and the receptor on the tumor cell surface.
This effect results in the increase of tumor antigens promoting an inflammatory process
that might activate a favorable immunological response 81
. Nevertheless, it has been
demonstrated that, for immunoliposomes against internalizing epitopes, drug release is
quicker than for non-internalizing ligands, leading to better and faster therapeutic efficacy
24. To achieve a similar intracellular drug concentration, non-internalizing ligands require
longer periods of time. Furthermore, on some occasions, those concentrations are not
totally attained.
Immunoliposomes are associated with favorable factors, such as the affinity or the strength
of the interaction between the mAb and antigen, and the multivalency or ability of mAbs to
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
40
attach to several antigen binding sites 82
. This multivalency may be induced by the
multimerization of antibodies or their fragments. This has important functional
implications, because these ligands augment the valency and avidity of the complex,
promoting a higher retention time on cell-surface receptors 83
. Indeed, it has been reported
that the use of multivalent antibodies to target liposomal formulations might also enhance
the internalization of tumor antigens and other cellular signals, such as the induction of
apoptosis and inhibition of cell growth 84,85
.
Another advantage for these immunoliposomes is that the internalization of encapsulated
drugs contributes to overcome tumor therapy resistance bypassing efflux pumps like P-
glycoproteins involved in the Multidrug Resistance effect (MDR) 7,86
. One example is the
transferrin targeted liposome encapsulating doxorubicin 55
which was able to increase drug
cytotoxicity 25 fold in resistant cells 87
.
4. PHARMACOKINETICS (PK) OF TARGETED LIPOSOMES
Targeted liposomes improve the selectivity of the drug release into the tumor area,
supporting an enhancement of drug efficacy 62
. To achieve appropriate tumor drug
concentrations, a prior evaluation of the pharmacokinetics (PK) of liposomal formulations
is highly recommended 62
.
PK advantages have been reported for liposomal formulations in comparison with the free
drug. Reduced body distribution, combined with a longer blood circulation time, in
particular for pegylated liposomes, translates into a lower volume of distribution (Vd) and
a higher Mean Residence Time (MRT) 6,56,88
. PK information about drug exposure
reflected in the half-life or area under the curve of drug plasma concentrations (AUC) is
often reported during preclinical evaluation of different types of liposomal formulations in
order to establish an increase in these parameters in relation to the free drug. However, it is
CHAPTER 1
41
difficult to perform an adequate comparison across formulations because PK properties are
highly dependent on liposome characteristics and stability. In line with this, particle size
and superficial charge play an important role. In the case of particle size, lower blood
clearance is associated with < 30 nm. However, these nanoparticles show an inefficient
tumor accumulation due to their higher renal excretion compared to bigger particle sizes 89
.
For liposomes with a particle size above 300 nm, these are mainly taken up by the liver and
spleen 90
. Therefore, the optimal range-size is between 80 and 150 nm 89,90
. For particle
charge, neutral liposomes present a lower RES effect compared to positively or negatively
charged liposomes, triggering longer circulating times 91,92
.
Thus, body drug disposition is drastically modified after pegylated liposome
administration, increasing the AUC, as occurs with Doxil. This presents 6 and 66 times
higher AUC than non-PEG and the free drug, respectively 93
.
In general, this higher drug availability is assumed to correlate with an enhancement in
tumor accumulation. There are many preclinical assays for studying these characteristics,
although they involve the use of healthy animals, as can be observed in Table 4. Here, the
differences between formulations are difficult to interpret due to the importance of the
tumor during PK characterization 94,95
.
On the one hand, in animal tumor models, circulation half-life for targeted nanocarriers is,
in general, shorter than for non-targeted carriers 96
. This difference might be explained by
the rapid receptor binding and complex internalization, which decrease the presence of the
formulation in blood. An interesting example has been reported for EGFR targeted
doxorubicin immunoliposomes administered to cancer patients. The half-life in blood was
31 h, whereas for non-targeted it was 55-70 h 96
. On the other hand, a linear PK has been
reported for pegylated liposomes, but in some cases, this linear PK may be combined with
a non-linear PK due to the specific binding to tumor cells. This is the case of liposomes of
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
42
paclitaxel. The drug released from the formulation binds to plasma proteins in a linear and
saturable manner 97
.
In other cases, stability is crucial because some liposomes present a biphasic plasma
concentration-time profile, which is characterized by an initial rapid clearance due to the
rapid drug release, followed by a slower elimination phase 93,98
. To these aspects, it has to
be added the lack of information regarding other important PK features, such as
distinguishing between encapsulated and released drug, liposome tumor distribution, drug
metabolites or immunogenic effects after repeated doses 93
. In addition, particularly for
immunoliposomes, ligand density is a factor directly related to their clearance rate as well
as their ability to enhance tumor drug delivery. Thus, higher ligand density is correlated
with higher elimination rate, as is observed in Table 5 99
.
Therefore, a balance between drug release and drug elimination would be required in order
to obtain those concentrations associated with an adequate response. As a result, the
pharmacokinetic and pharmacodynamic characterization of liposomal formulations should
be applied to optimize the in vivo behaviour of the formulation and facilitate the translation
of this information to patients 100
. However, there are few studies in this regard.
Table 4. PK of different liposomal formulations IV administered to tumor-free animals.
Type Formulation Encapsulated
molecule
EE
(%)
Particle
size
(nm)
Animal
model
Dose
(mg/kg)
Cmax
(mg/L)
AUC
last (h
µg/mL)
t1/2
(h)
CL
(mL/h) Ref.
LP Esphingomyelin: CH Vinblastine-N-
oxide (CPD100)
62.5 154
Female SW
30
0.05 0.06 5.50 8.90 127
LP
Esphingomyelin: CH:
DSPE-PEG 66.2 137 0.06
0.17
9.50 4.40
LP DSPC: DSPEPEG2000:
CH (TEA PN) Irinotecan
100 110
Female SD 10
N.A.
1407.80
6.80 7.10
128
LP
DSPC: DSPEPEG2000:
CH (TEA SOS) 100 110
2134.40
10.70 4.69
LP PC: CH Coumarin
98 105
Male SD 11.7
4.89 2.32 0.41 1263.75 129
T
PC: CH: DSPEPEG2000:
DSPEPEG2000-GAL 99 128 10.18 6.70 1.35 470.50
LP EPC: DOPE: CH Gemcitabine
15 177 Mice
0.45
N.A.
9.58
N.D. 54.30
130
T EPC: DOPE: CH 16 212 17.75 N.D. 26.70
LP: non-targeted liposome; T: Peptide-targeted liposome; SD: Sprague-Dawley rats; SW: Swiss Webster mice; N.A.: non-available. The harmonization of PK
parameters, some of them expressed by kg, has been done using the standard body weights, 25g for mice and 250g for rats.
Table 5. PK of different immunoliposomal formulations IV administered to tumor-free animals.
Type Formulation Encapsulated
molecule
EE
(%)
Particle
size (nm)
Animal
model
Dose
(mg/kg)
Cmax
(mg/L)
AUC
last (h
µg/mL)
t1/2
(h)
CL
(mL/h) Ref.
D Doxorubicin HCl
Doxorubicin
N.A.
---
SD 5
0.073 1.59 33.90 473.95
131 LP Doxil® 109 44.81 569.30 14.70 1.85
IL LP-Anti-CD147-DSPE-
PEG-Mal 91 42.13 240.96 15.80 4.13
D Daunorubicin
Daunorubicin
--- ---
SD 4 N.A.
7.40 1.72 107.75
125
LP S100PC: CH: mPEG2000-
DSPE 93 105 269.0 11.19 3.75
IL
Anti-CD123-Mal-
PEG2000-DSPE-
S100PC: CH:
mPEG2000-DSPE (1:400
mAb/LP)
91 113 114.3 7.61 8.00
IL
Anti-CD123-Mal-
PEG2000-DSPE-
S100PC: CH:
mPEG2000-DSPE (1:800
mAb/LP)
90 109 160.3 9.84 6.00
LP: non-targeted liposome; D: Active drug; IL: Immunoliposome; EE: Encapsulation efficacy; Cmax: Maximum serum concentration; AUC: Area under the curve; t1/2:
Half life ; CL: Clearance; N.A.: non available.
CHAPTER 1
45
It is clear that differences across species may limit the translational application of these
formulations. However, in an attempt to address this, more complex preclinical designs
seem to be necessary to cover the relevant properties of these formulations. The application
of physiological-based PK models which represent, in more mechanistic terms, liposomes
and drug disposition in the different organs, in particular, the tumor, spleen, and liver, may
help to yield preclinical and clinical findings and develop a better predictive model 97
.
5. CLINICAL TRIALS WITH TARGETED LIPOSOMES
Despite the advantages reported for targeted liposomes, few formulations have reached
clinical trials. In the present section, the status of the most promising targeted formulations,
including immunoliposomes, will be discussed.
5.1. Transferrin-targeted liposomes
MBP-426,
Transferrin (Tf) targeted liposomes encapsulating oxaliplatin, developed by Mebiopharm,
have demonstrated a greater therapeutic effect than previous oxaliplatin formulations 16
.
This drug presents limited antitumor effects due to a PK limitation derived from its high
levels of plasma proteins and erythrocyte binding that decrease free or therapeutic
concentrations. To overcome this, several preclinical studies have reported a better PK and
PD behavior following its encapsulation 48,106
.
Accordingly, the next step was liposome conjugation with Tf. This new formulation
demonstrated higher tumor selectivity than non-targeted liposomes and, consequently,
higher therapeutic activity. In a further optimization, lipid composition was modified by
incorporating NGPE, which changes its conformation in an acid pH. This effect leads to a
destabilization of both liposomes and lysosomes, promoting the intracellular release of
IMMUNOLIPOSOMES IN CLINICAL ONCOLOGY: STATE OF THE ART AND FUTURE
PERSPECTIVES
46
oxaliplatin and
Recommended