Human monoclonal antibodies

116 Pages·2010·6.53 MB·English

ETH Library
Human monoclonal antibodies
(mAbs)
applications in cancer and infectious disease
Doctoral Thesis
Author(s):
Sgier, David
Publication date:
2010
Permanent link:
https://doi.org/10.3929/ethz-a-006079450
Rights / license:
In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection.
For more information, please consult the Terms of use.
Diss. ETH N° 18856
Human monoclonal antibodies (mAbs): Applications in
cancer and infectious disease

A dissertation submitted to
ETH ZURICH
for the degree of
Doctor of Sciences

presented by
DAVID SGIER
Dipl. Pharm. Sciences ETH Zurich
Born on May 29, 1980
Citizen of Switzerland

Accepted on the recommendation of

Prof. Dr. Dario Neri, examiner
Prof. Dr. Karl-Heinz Altmann, co-examiner

2010
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To my parents
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INDEX

1 SUMMARY 9
2 ZUSAMMENFASSUNG 11
3 INTRODUCTION 13
3.1 Therapeutic human monoclonal antibodies (mAbs) 13
3.2 Generation of human monoclonal antibodies in vitro 21
3.2.1 Phage display 21
3.2.2 Alternative methodologies 27
3.2.2.1 Yeast surface display 27
3.2.2.2 Ribosome display 30
3.2.3 ETH-2-Gold library 33
3.2.4 In vitro affinity maturation 34
3.3 Urokinase-type plasminogen activator (uPA) as anti-cancer target 37
3.3.1 Physiological function of uPA 37
3.3.2 Urokinase in cancer 38
3.3.3 Drugs targeting uPA 39
3.4 Mycobacterium ulcerans infection: Buruli ulcer disease 41
3.4.1 History and epidemiology 41
3.4.2 Clinical presentation 43
3.4.3 Pathophysiology of M. ulcerans disease and the role of mycolactone 44
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3.4.4 Diagnosis 46
3.4.5 Management of M. ulcerans disease 47
4 RESULTS 48
4.1 Isolation and characterization of an inhibitory human monoclonal antibody
specific to the urokinase-type plasminogen activator uPA 48
4.1.1 Abstract 48
4.1.2 Introduction 49
4.1.3 Materials and Methods 52
4.1.3.1 Cell lines 52
4.1.3.2 Selection of antibodies from the ETH-2-Gold library by phage
display 52
4.1.3.3 Sequencing of scFv antibody genes 53
4.1.3.4 Characterization of scFv antibody fragments 53
4.1.3.5 Construction of affinity maturation libraries 53
4.1.3.6 Cloning and expression of IgG(DS2) 54
4.1.3.7 Surface Plasmon resonance (BIAcore) 54
4.1.3.8 UPA inhibition assay 55
4.1.3.9 Immunofluorescence (IF) on frozen tissue sections 55
4.1.3.10 Immunocytochemistry/confocal laser scanning microscopy 56
4.1.3.11 Tumor studies in mice 56
4.1.3.12 Ex vivo fluorescence experiments 57
4.1.4 Results 58
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4.1.4.1 Antibody phage display selections against uPA 58
4.1.4.2 In vitro characterization of scFv(DS2) and IgG(DS2) 60
4.1.4.3 Enzyme inhibition 62
4.1.4.4 Immunofluorescence analysis with IgG(DS2) 62
4.1.4.5 Therapeutic activity of IgG(DS2) against localized tumor growth 64
4.1.4.6 In vivo tumor targeting performance of IgG(DS2) 65
4.1.5 Discussion 71
4.2 Isolation and characterization of a human monoclonal antibody specific to
the Mycobacterium ulcerans derived toxin Mycolactone 74
4.2.1 Abstract 74
4.2.2 Introduction 75
4.2.3 Materials and Methods 78
4.2.3.1 Mycolactone analogues 78
4.2.3.2 Selection of antibodies from the ETH-2-Gold library by phage
display 78
4.2.3.3 Characterization of scFv antibody fragments 79
4.2.3.4 Construction of affinity maturation libraries 79
4.2.3.5 Cloning and expression of IgG(E7) 79
4.2.3.6 Surface Plasmon resonance (BIAcore) 80
4.2.3.7 Indirect immunofluorescence assay (IFA) 80
4.2.3.8 Immunohistochemistry (IHC) 81
4.2.4 Results 82
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4.2.4.1 Antibody phage display selections against the mycolactone analogue
PG-73 82
4.2.4.2 In vitro characterization of scFv(E7) and IgG(E7) 84
4.2.4.3 Application of scFv(E7) and IgG(E7) in direct staining of
Mycobacterium ulcerans cultures 86
4.2.5 Discussion 88
5 CONCLUSIONs 90
6 SUPPLEMENTARY MATERIAL 95
6.1 Nucleotide sequence of scFv(DS2) 95
6.2 Amino acid sequence of scFv(DS2) 95
6.3 Nucleotide sequence of IgG(DS2) 96
6.4 Amino acid sequence of IgG(DS2) 97
6.5 Nucleotide sequence of scFv(E7) 98
6.6 Amino acid sequence of scFv(E7) 99
6.7 Nucleotide sequence of IgG(E7) 99
6.8 Amino acid sequence of IgG(E7) 101
7 ACKNOWLEDGEMENTS 103
8 REFERENCES 104
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1 SUMMARY
Monoclonal antibodies (mAbs) have proven to be useful as human protein
therapeutics because they bind to drug targets with high affinity and specificity and
exhibit a favorable pharmacokinetic profile. Phage display is a powerful and widely
used technology for the isolation of mAbs. In this thesis we present the use of phage
display for the isolation of monoclonal antibodies for therapeutic applications in
cancer and infectious disease.
The first part of the thesis describes the generation and affinity maturation of a human
monoclonal antibody (termed DS2) against the human urokinase (uPA) capable of
inhibiting its enzymatic activity with an IC value in the low nanomolar range. The
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novel antibody cross-reacts with murine uPA. It was expressed both as scFv fragment
and in IgG format, allowing a systematic comparative immunofluorescence analysis
of the uPA expression patterns in a large panel of human and murine tumors and of
normal human tissue. While uPA was strongly expressed in virtually all tumor
specimens tested, it only exhibited a weak expression in certain normal tissues
(mainly in colon, lung, spleen and bone marrow). IgG(DS2) was not able to inhibit
cancer growth in immunocompromised mice bearing subcutaneous human MDA-
MB-231 or DoHH-2 tumors. However, an ex vivo immunofluorescence analysis
confirmed the ability of the DS2 antibody to preferentially localize at the tumor site
compared to normal organs. Collectively, these data suggest that uPA blocking
antibodies may not be indicated for cancer growth inhibition strategies, but may serve
as valuable tools for the implementation of pharmacodelivery strategies against a
variety of different tumors.
The second part of the thesis describes the isolation, affinity maturation and
characterization of a human monoclonal antibody (referred to as E7) specific to a
mycolactone analogue PG-73. Mycolactone is a toxin involved in Buruli ulcer, a
necrotizing skin disease caused by infection with Mycobacterium ulcerans.
Mycolactone is secreted by the bacteria and is considered to have cytotoxic and
immunosuppressive activities. Its mode of action is unclear, but in a guinea pig model
of the disease, purified mycolactone injected subcutaneously reproduces the natural
pathology, and mycolactone negative variants are avirulent, implying a key role for
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