Design and in vitro / in vivo evaluation of a targeted ...dadun.unav.edu/bitstream/10171/56360/1/Tesis_MerinoDiaz18.pdfComo no a mis amig@s, Sani, Ire, Car, Alex y Cris, sois y seréis

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.

REFERENCES

1. Quetglas JI, Hervas-Stubbs S, Smerdou C. The immunological profile of tumor-bearing

animals determines the outcome of cancer immunotherapy. Oncoimmunology.

2013;2(6):e24499. doi:10.4161/onci.24499.

2. Remesh A. Toxicities of anticancer drugs and its management. Int J Basic Clin Pharmacol.

2012;1(1):2. doi:10.5455/2319-2003.ijbcp000812.

3. Chatelut E, Delord J-P, Canal P. Toxicity patterns of cytotoxic drugs. Invest New Drugs.

2003;21(2):141-148. http://www.ncbi.nlm.nih.gov/pubmed/12889735.

INTRODUCTION

14

4. Merino M, Zalba S, Garrido MJ. Immunoliposomes in clinical oncology: State of the art and

future perspectives. J Control Release. 2018;275:162-176.

doi:10.1016/j.jconrel.2018.02.015.

5. Eloy JO, Claro de Souza M, Petrilli R, Barcellos JPA, Lee RJ, Marchetti JM. Liposomes as

carriers of hydrophilic small molecule drugs: strategies to enhance encapsulation and

delivery. Colloids Surf B Biointerfaces. 2014;123:345-363.

doi:10.1016/j.colsurfb.2014.09.029.

6. Tila D, Ghasemi S, Yazdani-Arazi SN, Ghanbarzadeh S. Functional liposomes in the

cancer-targeted drug delivery. J Biomater Appl. 2015;30(1):3-16.

doi:10.1177/0885328215578111.

7. Owens DE, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric

nanoparticles. Int J Pharm. 2006;307(1):93-102. doi:10.1016/j.ijpharm.2005.10.010.

8. Suk JS, Xu Q, Kim N, Hanes J, Ensign LM. PEGylation as a strategy for improving

nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev. 2016;99:28-51.

doi:10.1016/j.addr.2015.09.012.

9. Eloy JO, Petrilli R, Trevizan LNF, Chorilli M. Immunoliposomes: A review on

functionalization strategies and targets for drug delivery. Colloids Surf B Biointerfaces.

2017;159:454-467. doi:10.1016/j.colsurfb.2017.07.085.

10. Maeda H. Toward a full understanding of the EPR effect in primary and metastatic tumors

as well as issues related to its heterogeneity. Adv Drug Deliv Rev. 2015;91:3-6.

doi:10.1016/j.addr.2015.01.002.

11. Iyer AK, Khaled G, Fang J, Maeda H. Exploiting the enhanced permeability and retention

effect for tumor targeting. Drug Discov Today. 2006;11(17-18):812-818.

doi:10.1016/j.drudis.2006.07.005.

12. Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: The

key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul.

2001;41:189-207. doi:10.1016/S0065-2571(00)00013-3.

13. Haeri A, Zalba S, ten Hagen TLM, Dadashzadeh S, Koning GA. EGFR targeted

thermosensitive liposomes: A novel multifunctional platform for simultaneous tumor

targeted and stimulus responsive drug delivery. Colloids Surfaces B Biointerfaces.

2016;146:657-669. doi:10.1016/j.colsurfb.2016.06.012.

14. Kono K, Takashima M, Yuba E, et al. Multifunctional liposomes having target specificity,

temperature-triggered release, and near-infrared fluorescence imaging for tumor-specific

chemotherapy. J Control Release. 2015;216:69-77. doi:10.1016/j.jconrel.2015.08.005.

15. Guo P, You J-O, Yang J, Jia D, Moses MA, Auguste DT. Inhibiting metastatic breast cancer

cell migration via the synergy of targeted, pH-triggered siRNA delivery and chemokine axis

INTRODUCTION

15

blockade. Mol Pharm. 2014;11(3):755-765. doi:10.1021/mp4004699.

16. Li Q, Tang Q, Zhang P, et al. Human epidermal growth factor receptor-2 antibodies

enhance the specificity and anticancer activity of light-sensitive doxorubicin-labeled

liposomes. Biomaterials. 2015;57:1-11. doi:10.1016/j.biomaterials.2015.04.009.

17. Sapra P, Shor B. Monoclonal antibody-based therapies in cancer: advances and challenges.

Pharmacol Ther. 2013;138(3):452-469. doi:10.1016/j.pharmthera.2013.03.004.

18. Merelli B, Massi D, Cattaneo L, Mandalà M. Targeting the PD1/PD-L1 axis in melanoma:

biological rationale, clinical challenges and opportunities. Crit Rev Oncol Hematol.

2014;89(1):140-165. doi:10.1016/j.critrevonc.2013.08.002.

19. Mahoney KM, Rennert PD, Freeman GJ. Combination cancer immunotherapy and new

immunomodulatory targets. Nat Rev Drug Discov. 2015;14(8):561-584.

doi:10.1038/nrd4591.

20. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity.

2013;39(1):1-10. doi:10.1016/j.immuni.2013.07.012.

21. Dunn GP, Old LJ, Schreiber RD. The immunobiology of cancer immunosurveillance and

immunoediting. Immunity. 2004;21(2):137-148. doi:10.1016/j.immuni.2004.07.017.

22. Chang C-H, Qiu J, O’Sullivan D, et al. Metabolic Competition in the Tumor

Microenvironment Is a Driver of Cancer Progression. Cell. 2015;162(6):1229-1241.

doi:10.1016/j.cell.2015.08.016.

23. Contreras-Sandoval AM, Merino M, Vasquez M, Trocóniz IF, Berraondo P, Garrido MJ.

Correlation between anti-PD-L1 tumor concentrations and tumor-specific and nonspecific

biomarkers in a melanoma mouse model. Oncotarget. 2016;7(47):76891-76901.

doi:10.18632/oncotarget.12727.

24. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to

activate anti-tumor immunity. Curr Opin Immunol. 2012;24(2):207-212.

doi:10.1016/j.coi.2011.12.009.

25. Robert C, Soria J-C, Eggermont AMM. Drug of the year: programmed death-1

receptor/programmed death-1 ligand-1 receptor monoclonal antibodies. Eur J Cancer.

2013;49(14):2968-2971. doi:10.1016/j.ejca.2013.07.001.

26. Blank CU. The perspective of immunotherapy: new molecules and new mechanisms of

action in immune modulation. Curr Opin Oncol. 2014;26(2):204-214.

doi:10.1097/CCO.0000000000000054.

27. Borch TH, Donia M, Andersen MH, Svane IM. Reorienting the immune system in the

treatment of cancer by using anti-PD-1 and anti-PD-L1 antibodies. Drug Discov Today.

2015;20(9):1127-1134. doi:10.1016/j.drudis.2015.07.003.

28. Philips GK, Atkins M. Therapeutic uses of anti-PD-1 and anti-PD-L1 antibodies. Int

INTRODUCTION

16

Immunol. 2015;27(1):39-46. doi:10.1093/intimm/dxu095.

29. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature.

2011;480(7378):480-489. doi:10.1038/nature10673.

30. Shi T, Ma Y, Yu L, et al. Cancer Immunotherapy: A Focus on the Regulation of Immune

Checkpoints. Int J Mol Sci. 2018;19(5). doi:10.3390/ijms19051389.

31. Casares N, Pequignot MO, Tesniere A, et al. Caspase-dependent immunogenicity of

doxorubicin-induced tumor cell death. J Exp Med. 2005;202(12):1691-1701.

doi:10.1084/jem.20050915.

32. Tesniere A, Apetoh L, Ghiringhelli F, et al. Immunogenic cancer cell death: a key-lock

paradigm. Curr Opin Immunol. 2008;20(5):504-511. doi:10.1016/j.coi.2008.05.007.

33. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy.

Annu Rev Immunol. 2013;31:51-72. doi:10.1146/annurev-immunol-032712-100008.

34. Locher C, Conforti R, Aymeric L, et al. Desirable cell death during anticancer

chemotherapy. Ann N Y Acad Sci. 2010;1209:99-108. doi:10.1111/j.1749-

6632.2010.05763.x.

35. Emens LA. Chemoimmunotherapy. Cancer J. 16(4):295-303.

doi:10.1097/PPO.0b013e3181eb5066.

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: [email protected] (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

.


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


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

.


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


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.


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


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


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

.


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


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


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


PERSPECTIVES

46

oxaliplatin and

Documents

Design and in vitro / in vivo evaluation of a targeted ...dadun.unav.edu/bitstream/10171/56360/1/Tesis_MerinoDiaz18.pdfComo no a mis amig@s, Sani, Ire, Car, Alex y Cris, sois y seréis