Intracellular delivery of therapeutic antibodies. PhD thesis, University of Nottingham.

285 Pages·2017·8.45 MB·English

by  Christine Middleton

Intracellular delivery of therapeutic
antibodies

By Gavin S. Hackett, BSc (Hons)

Thesis submitted to the University of Nottingham for the degree
of Doctor of Philosophy

October 2011
Abstract

Therapeutic antibodies are highly versatile macromolecules that can be engineered to
bind and inhibit a target with high specificity. Unfortunately, the cell membrane is
impenetrable to antibody reagents, thus limiting their use almost entirely to
extracellular targets. Expanding the application of therapeutic antibodies to
intracellular targets is an exciting concept that could have a huge impact on how
intracellular protein-protein interactions involved in diseases can be modulated. The
modification of therapeutic antibodies with Cell Penetrating Peptides (CPPs) can
enable cellular penetration, however, no general approach to modifying antibodies
with CPPs has been developed that allows for systematic optimisation of both the in
vivo and cell penetrating properties.
In this study, neutralising single-chain variable fragment antibodies (scFvs) have been
isolated from naïve scFv libraries using antibody phage display that are specific to the
model intracellular targets Bcl-2 and Bcl-xL. Lead scFvs showed potent inhibition of
these proteins in an in vitro assay with IC values of <10 nM being calculated, which
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is superior to the small molecule Bcl-2/xL inhibitor ABT-737. The lead anti-Bcl-xL
scFv was conjugated to the CPPs octa-arginine, HIV Tat or Antp . These
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peptides were synthesised to possess either an N-isobutyryl cysteinyl or N-
maleimidopropionyl moiety and a C-terminal lysine residue, allowing for site-specific
conjugation to an unpaired cysteine residue introduced to the scFv construct and
regioselective introduction of 5-carboxyfluorescein to the CPP, respectively. Live-cell
confocal microscopy showed that the scFv-octa-arginine conjugate possessed superior
cell entry capabilities compared to the scFv-Tat and scFv-Antp conjugates.
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Further studies using a panel of cancer cell lines are required to determine if the anti-
Bcl-xL scFv-octa-arginine conjugate can induce apoptosis through inhibition of
cellular Bcl-xL.
Additonally, a novel approach to controlling the cell penetrating properties of the CPP
octa-arginine has been developed. It was demonstrated that carbamate protection of
octa-arginine’s guanidine functionality effectively inhibited its cell entry capabilities.
Moreover, esterase-labile acyloxymethyl carbonyl (AM) protecting groups were

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utilised to protect the guanidine functionality of octa-arginine, inhibiting its cell entry
capabilities. In a HPLC based assay it was demonstrated that the AM protected octa-
arginine was deprotected by pig liver esterase, suggesting that in vivo deprotection
could be achieved by serum esterases. The controlled unmasking of octa-arginine is
predicted to increase its circulation time and reduce non-specific tissue uptake in vivo,
potentially making this CPP more suitable for in vivo applications.
The methodologies utilised for the preparation of scFv-CPP conjugates and developed
for the controlled unmasking of octa-arginine in this study will allow for optimisation
of the cell penetrating and in vivo properties of this promising class of macromolecular
therapeutic, thus providing a gate-way to unlocking the immense potential of
therapeutic intracellular antibodies.

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Acknowledgments

I would like to thank my supervisors Peter Fischer, Weng Chan and Ron Jackson for
their continued support, encouragement and guidance throughout my PhD studies. I
would also like to acknowledge the EPSRC, AstraZeneca and MedImmune for
funding the PhD.
I am especially grateful to all of the present and past members of the chemistry
corridor in CBS for their friendship and for providing an enjoyable working
atmosphere. I particularly want to thank Cillian Byrne, Chris Gordon, Charlie
Mathews and Chou-Hsiung Chen for their invaluable suggestions and help. I also
want to thank Chris for his encouragement while I was writing-up and for proof-
reading my thesis.
I am extremely grateful for all of the help and invaluable advice from so many people
at MedImmune in Cambridge, in particular Monica Papworth, Peter Ravn, Siobhan
O'Brien, Jeff Revell, Anna-Lena Schinke, Des O'Shea and Sara Carmen for both
advice with my work and also friendship, making the time that I spent in Cambridge
an enjoyable and invaluable experience.
I would also like to express my thanks to all of the technical staff in CBS, in particular
Lee Hibbett and Graham Coxhill for help with the HPLC, MS and many other lab
related things.
I must also thank my two past house-mates Asa Bluck and Graham Mullard for always
being ready for a beer (or two…) after work. Finally, I thank my parents, brothers,
sister, and grandparents for supporting me when things were difficult. I am also very
grateful to Jessica Chu for her support throughout the final year of my PhD studies .

This thesis is dedicated to my parents

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Abbreviations

ACPP Activatable cell penetrating peptide
Alpha screen Amplified luminescent proximity homogeneous assay
AM Acyloxymethyl carbonyl
AMI Acute myocardial infarction
Antp Penetratin
AO Acyloxy carbonyl
ATTEMPTS Antibody targeted, [protamine] triggered, electrically modified
pro-drug strategy
BH 1- 4 Bcl-2 homology domain 1-4
CDR Complementarity determining region
CHO Chinese hamster ovary
CPP Cell penetrating peptide
EC 50% maximal effective concentration
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ELISA Enzyme linked immunosorbant assay
Fab Fragment antigen binding region
FBS Fetal bovine serum
Fc region Fragment crystalline
Fv Variable domain
HIV Human immunodeficiency virus
HPLC High-performance liquid chromatography

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HRMS High resolution mass mass spectrometry
HRP Horse radish peroxidase
HSPG Heperan sulfate proteoglycans
HTS High throughput screening
IACT Intracellular antibody capture technology
IC 50% maximal inhibitory concentration
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%ID/g % Injected dose per gram of target tissue
K Dissociation constant
d
K Inhibition constant
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MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight
MMP Matrix metalloproteinase
MS Mass spectrometry
naCPP Non-amphipathic cell penetrating peptide
NK1R Neurokinin-1 receptor
NMR Nuclear magnetic resonance
paCPP Primary amphipathic cell penetrating peptide
PD Pharmacodynamic
PLE Pig liver esterase
PK Pharmacokinetic
PPI Protein-protein interaction
R Retention factor
f
RISC RNA-induced silencing complex
RNAi RNA interference

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rt Room tempertaure
SA Streptavidin
saCPP Secondary amphipathic cell penetrating peptide
ScFv Single chain variable fragments
SMoC Small molecule carrier
SPPS Solid-phase peptide synthesis
Tat HIV Transactivator of Transcription
TNF Tumour necrosis factor
TOF Time-of-flight

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Contents

1 Introduction .......................................................................................................................................... 1
1.1 Inhibiting protein-protein interactions ..................................................................................... 1
1.2 Therapeutic antibodies ............................................................................................................. 4
1.2.1 Antibody Engineering ..................................................................................................... 5
1.3 Targeting intracellular protein-protein interactions with antibodies ........................................ 9
1.3.1 Intrabodies .................................................................................................................... 11
1.3.2 Transbodies ................................................................................................................... 13
1.4 Cell penetrating peptides ....................................................................................................... 16
1.4.1 Mechanism of CPP internalisation ................................................................................ 19
1.4.2 The application of CPPs in vivo .................................................................................... 22
1.4.3 Modulation of CPP in vivo properties ........................................................................... 25
1.5 Making Transbodies .............................................................................................................. 30
1.5.1 Chemical conjugation of CPPs to antibodies ................................................................ 31
1.6 Research aims and objectives ................................................................................................ 39
2 Isolation of neutralising single chain antibodies specific to Bcl-2 .................................................. 42
2.1 Bcl-2 - The model target ........................................................................................................ 42
2.2 Isolation of anti-Bcl-2 and Bcl-xL scFv using antibody phage display ................................. 46
2.2.1 Selection of anti-Bcl-2 and anti-Bcl-xL scFvs .............................................................. 49
2.3 Identification of scFv neutralisating towards Bcl-2 and Bcl-xL ............................................ 54
2.3.1 Profiling of positive hits ............................................................................................... 59
2.3.2 Binding of scFv in reducing environment ..................................................................... 62
2.4 Conclusions ............................................................................................................................ 64
2.5 Materials and Methods ........................................................................................................... 66
2.5.1 Biotinylation of Bcl-2 and Bcl-xL ................................................................................ 66
2.5.2 Direct binding ELISA ................................................................................................... 67
2.5.3 Phage selection protocol ............................................................................................... 68
2.5.4 Phage rescue for subsequent selection rounds .............................................................. 70
2.5.5 Analysis of selected phage ............................................................................................ 70
2.5.6 Phage ELISA ................................................................................................................ 71
2.5.7 High-throughput screening ........................................................................................... 72
2.5.8 Alpha Screen profiling of His-tag affinity purified scFv. ............................................. 74
2.5.9 scFv binding ELISA in the presence of reduced glutathione ........................................ 74
3 Preparation and characterisation of scFv-CPP conjugates ............................................................ 76
3.1 Synthetic approaches for scFv-CPP conjugation ................................................................... 77
3.2 Expression of scFv-Cys ......................................................................................................... 78

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3.2.1 Mammalian expression of scFv-Cys ............................................................................. 82
3.3 Fmoc solid-phase peptide synthesis of CPPs ......................................................................... 87
3.3.1 Synthetic strategies for CPP derivatives ....................................................................... 89
3.3.2 Penetratin derivatives .................................................................................................... 92
3.3.3 Octa-arginine derivatives .............................................................................................. 98
3.3.4 HIV Tat derivatives ............................................................................................... 100
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3.3.5 Pro derivatives .......................................................................................................... 102
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3.3.6 Summary of CPP derivative synthesis ........................................................................ 104
3.4 Preparation of scFv-CPP conjugates ................................................................................... 105
3.4.1 Thioether conjugation ................................................................................................. 105
3.4.2 Disulfide conjugation .................................................................................................. 107
3.4.3 Summary of scFv-CPP conjugate preparation ............................................................ 111
3.5 Cellular delivery of scFv-CPP(FAM) conjugates ................................................................ 114
3.5.1 Fluorescence microscopy ............................................................................................ 114
3.5.2 Confocal Laser Scanning Microscopy (CLSM) .......................................................... 116
3.5.3 Summary of scFv-CPP cell internalisation ................................................................. 121
3.6 Biological activity of scFv-CPP conjugates ........................................................................ 122
3.6.1 The effect of scFv-CPP constructs on Rat basophilic leukaemia cell (RBL-2H3)
viability .................................................................................................................................... 124
3.7 Conclusions ......................................................................................................................... 126
3.8 Materials and Methods ........................................................................................................ 129
3.8.1 SDS-PAGE and western blot analysis ........................................................................ 129
3.8.2 General sub-cloning procedure ................................................................................... 130
3.8.3 Expression of scFv-Cys in E. coli with pUC119MCH vector .................................... 132
3.8.4 Expression of scFv-Cys in E. coli with p10HISCYS vector ....................................... 133
3.8.5 Transient expression of scFv-Cys in Cep6 cells with pEOMCH vector ..................... 135
3.8.6 Preparation of scFv-CPP conjugates ........................................................................... 137
3.8.7 General cell culture protocol ....................................................................................... 139
3.8.8 Fluorescence microscopy ............................................................................................ 140
3.8.9 Confocal scanning laser microscopy........................................................................... 140
3.8.10 Biological activity of scFv-CPP conjugates - Cell Titre Glo assay® .................. 141
4 Isolation of neutralising single chain antibodies specific to Bcl-2 ................................................ 143
4.1 Proof-of-concept: Inhibition of CPP cell internalisation by carbamate protection of the
guanidine moieties ............................................................................................................................ 146
4.1.1 Cell internalisation of protected octa-arginine derivatives ......................................... 157
4.2 Protection of arginine side-chain with hydrolytic and esterase-labile protecting groups .... 161
4.2.1 Synthesis of protected isothioureas ............................................................................. 164
4.2.2 The stability of acyloxycarbonyl and acyloxmethyoxy carbonyl protected arginine .. 169
4.2.3 Synthesis of an acyloxymethyl carbonyl protected octa-arginine derivative .............. 184

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4.1 Conclusions ......................................................................................................................... 192
4.2 Materials and methods ......................................................................................................... 195
4.2.1 Hydrolytic stability assay ............................................................................................ 195
4.2.2 Esterase assay ............................................................................................................. 195
4.2.3 Confocal scanning laser microscopy........................................................................... 196
5 Conclusions and future directions .................................................................................................. 197
6 Experimental .................................................................................................................................... 210
6.1 Materials and Instrumentation ............................................................................................. 210
6.1.1 General method for Fmoc solid-phase peptide synthesis of CPPs .............................. 211
6.2 Experimental for Chapter 3 ................................................................................................. 213
6.3 Experimental for Chapter 4 ................................................................................................. 224
7 References ......................................................................................................................................... 243
Appendix I-III…………………………………………………………………………………………263

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