Chapter 6 Incorporation of unnatural amino acids in nanobodies

160 Pages·2014·10.24 MB·English

The printing of this thesis was financially supported by:
Amgen B.V.
INstruchemie B.V.
Macrogen Europe
Pfizer B.V.
Colofon
ISBN: 978-94-6295-001-6
Cover design: Remko van Vught
Printed by: Proefschriftmaken.nl || Uitgeverij BOXPress
Published by: Uitgeverij BOXPress, ‘s-Hertogenbosch
The research presented in this thesis was performed at the Membrane Biochemistry
and Biophysics group of the Utrecht University. This work was funded by the Focus &
Massa project of the Utrecht University, The Netherlands.
Copyright © Remko van Vught 2014, Utrecht, The Netherlands
All rights reserved. No part of this thesis may be reproduced, stored in a retrieval
system, or transmitted in any form or by any means, without written permission of the
author and the publisher holding the copyrights of the published articles.
Functionalized nanobodies
for cancer therapy
Gefunctionaliseerde nanobodies als kankertherapie
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit Utrecht op
gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge
het besluit van het college voor promoties in het openbaar te verdedigen
op woensdag 26 november 2014 des middags te 2.30 uur
door
Remko Wilhelmus Maria van Vught
geboren op 28 februari 1986
te Tilburg
Promotoren: Prof.dr. J.A. Killian
Prof.dr. R.J. Pieters
Copromotor: Dr. E.J. Breukink
Voor mijn ouders

Table of contents
Chapter 1 General introduction 9

Chapter 2 Site-specific functionalization of proteins and their 15
applications to therapeutic antibodies

Chapter 3 Nanobodies and their therapeutic applications 41

Chapter 4 Intein-mediated functionalization of nanobodies 57

Chapter 5 Epidermal growth factor receptor-targeted liposomes 77
with zinc phthalocyanine for photodynamic therapy

Chapter 6 Incorporation of unnatural amino acids in nanobodies 97

Chapter 7 Selective targeting of EGFRvIII-positive glioblastomas 119
by a nanobody-based immunotoxin

Chapter 8 Summarizing discussion 139

Addendum Nederlandse samenvatting 147
Curriculum Vitae 151
List of publications 153
Dankwoord 155
1
Chapter 1

General introduction
Chapter 1
Cancer treatment
Cancer is the collective term for diseases associated with uncontrolled cell division. In 2012, cancer
was estimated to be diagnosed in 14.1 million people worldwide and the cause of 8.2 million
deaths1. The development of cancer has been attributed to genetic (5 - 10 %) and environmental
factors (90 – 95 %)2. Environmental factors can be subdivided into the environment and lifestyle,
1
of which the latter is the main cause of cancer-related deaths and includes smoking, diet and
infections. The development of cancer requires 4-6 mutations to transform normal cells into
cancer cells3. In this process, mechanisms regulating proliferation are lost and cells become self-
1
sufficient in proliferative signals.
The line of cancer treatment depends on the disease state, aggressiveness and the
timing of its discovery. Where surgical interference is often applied for solid tumors, radio- and
chemotherapy is used for smaller tumors or metastases and also to treat residuals after surgery.
To distinguish cancer cells from healthy cells, most chemotherapeutic drugs target rapidly
growing cells by acting on DNA replication or mitosis. Unfortunately, these cytotoxic drugs
also target cell types with natural rapid cell division including cells in the digestive tract, bone
marrow, and hair follicles, and thus give rise to (severe) side effects.
Targeted therapies
A new class of anti-cancer drugs, the targeted therapies, aims to minimize these side effects
by acting on one specific molecule. Targeted therapies can be divided into two groups: small
molecule inhibitors (mainly tyrosine-kinase inhibitors) and monoclonal antibodies (mAbs)4. As
tyrosine kinases are involved in signal transduction, theses small molecules prevent activation
of the receptor and subsequently inhibit proliferation5. In contrast, mAbs are restricted to bind
molecular targets on the outside of the cell and act on multiple levels: 1) blocking receptor
activation by either binding the receptor at the ligand-receptor interface or by binding the ligand
itself, 2) activating antibody dependent cell-mediated cytotoxicity, 3) inducing complement
dependent cytotoxicity6. Their high specificity and safety profile led to the development of mAbs
for various cancer indications7. A new generation of mAbs are the antibody drug conjugates
(ADCs), which deliver very potent drugs to target cells and thereby limit side effects8.
These conventional IgG antibodies are 150 kDa and bear two heavy and light chains
(Fig. 1). As a consequence of their size (and recycling via the neonatal Fc receptor) mAbs have
a long half-life and therapeutic effect9. In contrast, however, the tumor penetration of mAbs is
hampered by the size10. This led to the development of antibody fragments, which are smaller
without affecting the specificity. One promising set of antibody fragments are derived from
heavy-chain antibodies in camelids (Fig. 1). These antibody fragments are only 15 kDa and
known as nanobodies or VHH (Variable domains derived from the Heavy chain of Heavy chain
only antibodies). Next to their small size, nanobodies possess unique features when compared
10