L19 antibody conjugates in cancer and

163 Pages·2008·6.97 MB·Italian

ETH Library
L19 antibody conjugates in cancer
and cardiovascular disease
Doctoral Thesis
Author(s):
Alessi, Patrizia
Publication date:
2004
Permanent link:
https://doi.org/10.3929/ethz-a-004907799
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.ETHNr. 15764
L19 antibody conjugates in and
cancer
cardiovascular disease
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the degree of
DOCTOR OF NATURAL SCIENCES
Presented by
PatriziaAlessi
Dottore in Biotecnologie Agro-Industriali University ofVerona
-
born January 08, 1975
from Verona, Italy
accepted on the recommendation of
Prof. Dr. DarioNeri, examiner
Prof. Dr. Karl-Heinz Altmann, co-examiner
Zürich, October2004
/
Seite Leer
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"Beato chi trova in te lasuaforza
e decide nelsuo cuore ilsanto viaggio"
(Salmo83,6)
A Fra Massimo, Madre Miriam
Mimmo e Cinzia
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TABLE OF CONTENTS
SUMMARY SOMMARIO 11
-
INTRODUCTION 19
2.1 Molecular targeting ofangiogenesis 21
2.1.1 Angiogenesis and angiogenesis-related disease 23
2.1.2 Angiogenesis and Atherosclerosis 26
2.1.3 Moleculartargetinfofangiogenesis: a definition 29
2.1.3.1 Markers ofangiogenesis for ligand-based
vasculartargrting strategies 29
2.1.3.2 Integrins avß3 and avß5 30
2.1.3.3 Prostatic-specific membrane antigen (PMSA) 31
2.1.3.4 Endoglin (CD105) 32
2.1.3.5 VEGF VEGF-receptors complex 32
2.1.3.6 CD44 33
2.1.3.7 Phosphatidyl serine phospholipids 34
2.1.3.8 Large isoforms oftenascin C 34
2.1.3.9 Magic roundabout 35
2.1.3.10 Others markers 35
2.1.4 ED-B domain offibronectin 36
2.1.5 The L19 antibody and its recombinant formats 39
2.1.6 Some considerations on vasculartargeting antibodies 40
2.1.7 Positron Emission Tomography (PET) and near Infrared
(NIR) Fluorescence imaging modalities 44
2.2 Antibodies-drug conjugates in cancer therapy 49
2.3 Antibody -enzyme conjugates for produrg therapy 60
3. RESULTS 67
3.1 Antibody production 69
3.2 Molecular imaging ofangiogenesis in cancer 73
3.2.1 In vivo imaging with 1241-SIP(L19) by Positron Emission
Tomography (PET) 73
3.2.2 Ex vivo imaging: targeting a malignantglioma in rat 75
3.3 Molecular imaging ofatherosclerotic plaques using SIP(L19) 78
3.3.1 Labeling ofantibodies with radioiodine or fluorescent dyes 78
3.3.2 SIP(L19) selectively targets murine plaques 78
3.3.3 Cy7-labeled SIP(L19) allows macroscopic fluorescence
detection ofmurine plaques 83
3.3.4 The ED-B domain offibronectin is overexpressed in
murine and human plaques 85
3.4 Selective occlusion oftumor blood vessels by targeted delivery
ofan antibody-photosensitizer conjugate 86
3.4.1 Photosensitizer-antibody conjugation 86
3.4.2 In vivo PDT experiments 86
3.5 L19-doxorubicin conjugates 89
3.5.1 Design and synthesis ofa SIP(L19)-doxorubicinconjugates 90
3.5.2 Therapeutic properties ofSIP(L19)-doxorubicinconjugates
in tumor- bearing mice 93
3.5.3 Modification ofantibody carbohydrates by oxidation and
conjugation with Doxorubicin 96
3.5.4 Kinetic analysis ofthe released Doxorubicin from antibody
conjugates 99
3.5.5 In vivo anti-tumorefficacy ofIgGl(L19)-doxorubicin 100
3.6 Antibody-enzyme conjugate: coupling human prolyl endopeptidase
to SIP(L19) antibody 104
3.6.1 Enzymatic hydrolysis and cytotoxic activity ofprodrugs 104
3.6.2 Synthesisofimmunoconjugate 107
4. MATERIAL AND METHODS 109
4.1 Antibody preparation 111
4.1.1 Cloning ofSIP(HyHEL-10) 111
4.1.2 Expression andpurification ofS1P(L19), SIP(HyHEL-lO) and
IgGl(L19) 111
4.2 RadioimmunoconjugatesofL19 antibody 112
4.2.1 Coupling 124I to SIP(L19) 112
4.2.2 Coupling 131I to SIP(L19) 113
4.3 Antibody-based plaque targeting 114
4.3.1 Coupling radioisotopes and fluorophores to SIP(L19) and
SIP(HyHEL-lO) 114
4.3.2 Animals 115
4.3.3 Harvestand tissue processing 115
4.3.4 Tissue analysis 115
4.3.5 Statistics 116
4.4 Vasculartargetingwith antibody-photosensitizer conjugates 116
4.4.1 Cell lines 116
4.4.2 Animals, tumor induction and PDT protocols 117
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4.5 L19-Doxorubicin conjugates 118
4.5.1 Synthesis ofantibody-Doxorubicin conjugates obtained by
oxidation with sodium periodate 118
4.5.1.1 Direct coupling between SIP(L19) and Doxorubicin-
derivative 118
4.5.1.2 Modification ofmonoclonal antibody carbohydreates
by oxidation and conjugationwithDoxorubicin 119
4.5.2 Release experiment ofDoxorubicin from from
IgGl(L19)-doxorubicin immunoconjugate 120
4.5.3 In vivo tumor studies 120
4.5.4 Biodistribution experiment 121
4.6 Prolyl endopeptidase for ADEPT 121
4.6.1 Chemical synthesis ofthe prodrugs 121
4.6.2 Prodrug activation by prolyl endopeptidase 122
4.6.3 Cytotoxicity assays 122
4.6.4 Synthesis ofthe immunoconjugate 123
DISCUSSION 125
REFERENCES 131
ACKNOWLEDGEMENTS 153
CURRICULUM VITAE ET STUDIORUM 157
The abbreviations used are: ED-B, extra domain B offibronectin; scFv, single chain Fv
antibody fragment; LI9, human monoclonal antibody specific to ED-B; mAb, monoclonal
Antibody; NHS, N-hydroxysuccinimide; SIP(L19), recombinant miniantibody (Small
ImmunoProtein) format of LI9; SIP(HyHEL-lO), recombinant miniantibody HyHEL-10
(directed against hen egg lysozyme); ApoE7", Apolipoprotein E null mice; Cy5 and Cy7,
commercially avaible fluorophores; PET, Positron Emission Tomography; PBS, Phosphate
buffered saline; PDT, Photodynamic Therapy; SDS-PAGE, sodium dodecyl sulfate
Polyacrylamide electrophoresis; SnChe6, bis (triethanolamine)Sn(IV)chlorin e6; PEP
(prolyl endopeptidase), ADEPT (antibody directed enzyme prodrug therapy), AMC (7-
amino-4-methyl-coumarin), Z (benzyloxycarbonyl), CHO (chinese hamster ovary), MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide), PEG (polyethylene
glycol), MBS (m-Maleimidobenzoyl-N-hydroxysuccinimideester).
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