IMMUNOTHERAPY FOR ALZHEIMER'S DISEASE

208 Pages·2017·7.33 MB·English

ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús
establertesperlasegüentllicènciaCreativeCommons: http://cat.creativecommons.org/?page_id=184
ADVERTENCIA.Elaccesoaloscontenidosdeestatesisquedacondicionadoalaaceptacióndelascondicionesdeuso
establecidasporlasiguientelicenciaCreativeCommons: http://es.creativecommons.org/blog/licencias/
WARNING.Theaccesstothecontentsofthisdoctoralthesisitislimitedtotheacceptanceoftheuseconditionsset
bythefollowingCreativeCommonslicense: https://creativecommons.org/licenses/?lang=en
Departament de Bioquímica i Biologia Molecular

IMMUNOTHERAPY FOR
ALZHEIMER’S DISEASE:
from antibody engineering to
combined therapy with
apolipoproteins.

Doctoral Thesis

Laia Montoliu Gaya

2017

Departament de Bioquímica i Biologia Molecular

IMMUNOTHERAPY FOR
ALZHEIMER’S DISEASE:
from antibody engineering to
combined therapy with
apolipoproteins.

Doctoral Thesis presented by Laia Montoliu Gaya
for the Degree of Ph.D in Biochemistry, Molecular
Biology and Biomedicine, supervised by Dra.
Sandra Villegas.

Laia Montoliu Gaya Sandra Villegas

Bellaterra, June 2017

CONTENTS

TABLE OF CONTENTS ................................................................................................. i
LIST OF FIGURES AND TABLES .................................................................................. iv
ABBREVIATIONS .....................................................................................................viii
ABSTRACT ................................................................................................................ x

I-INTRODUCTION ...................................................................................................... 1
1. ALZHEIMER’S DISEASE .................................................................................................. 3
2. AMYLOID CASCADE HYPOTHESIS ................................................................................. 3
2.1 Aβ generation by the γ-secretase complex ............................................................... 5
2.2 Amyloid-β (Aβ) peptide ............................................................................................. 5
3. GENETICS OF AD ........................................................................................................... 7
4. APOLIPOPROTEINS AND AD ......................................................................................... 8
4.1 Apolipoprotein E (APOE) ........................................................................................... 8
4.2 Apolipoprotein J/Clusterin (APOJ/CLU) ................................................................... 11
5. IMMUNOTHERAPY ..................................................................................................... 12
5.1 Active immunotherapy ............................................................................................ 12
5.2 Passive immunotherapy .......................................................................................... 13
5.2.1 Mabs: Bapineuzumab (AAB-001 & AAB-003, Pfizer/Janssen) .................. 15
5.2.2 Single-chain variable fragment (ScFv) ...................................................... 16
6. ScFv-h3D6 .................................................................................................................. 17
6.1 ScFv-h3D6 characterization ...................................................................................... 17
6.2 In vivo validity of scFv-h3D6 .................................................................................... 21
6.3 Improvement of scFv-h3D6 stability ........................................................................ 23
6.4 The scrambling problem .......................................................................................... 24

II-GENERAL AIMS .................................................................................................... 25

III-GENERAL METHODS ........................................................................................... 29
1. RECOMBINANT PROTEIN PRODUCTION ........................................................................... 31
i
1.1 Polymerase Chain Reaction (PCR) ................................................................................ 31
1.2 Protein Expression ........................................................................................................ 32
1.3 Protein Purification ...................................................................................................... 34
2. PROTEIN CHARACTERIZATION ......................................................................................... 35
2.1 Circular Dichroism (CD) ................................................................................................ 35
2.2 Fluorescence Spectroscopy .......................................................................................... 36
2.3 Fourier Transformed Infrared Resonance (FTIR) .......................................................... 38
2.4 Transmission Electron Microscopy (TEM) .................................................................... 40
2.5 Differential Scanning Calorimetry (DSC) ...................................................................... 40
2.6 Mass spectrometry ....................................................................................................... 42
3. CELL CULTURE .................................................................................................................. 43
3.1 SH-SY5Y neuroblastoma cell line .................................................................................. 43
3.2 Viability assays ............................................................................................................. 43
3.3 Human primary astrocyte cell culture .......................................................................... 44
3.4 Flow cytometry ............................................................................................................. 44
3.5 Enzyme-linked immunoabsorbent assay (ELISA) ......................................................... 45
4. IN VIVO STUDIES .............................................................................................................. 46
4.1 Mice 3xTg-AD ................................................................................................................ 46
4.2 Enzyme-linked immunoabsorbent assay (ELISA) ......................................................... 47
6.3 Immunohistochemistry ................................................................................................. 47

IV-CHAPTERS .......................................................................................................... 49

CHAPTER 1: Understanding the contribution of disulphide bridges to the folding and
misfolding of scFv-h3D6 ........................................................................................... 51
Aims ........................................................................................................................ 53
Materials and Methods ......................................................................................... 54
Results .................................................................................................................... 55
Discussion ............................................................................................................... 65
CHAPTER 2: Towards the improvement in stability of scFv-h3D6 as a way to enhance
its therapeutic potential ........................................................................................ 67
Aims ........................................................................................................................ 69
Materials and Methods ......................................................................................... 70
Results and Discussion ........................................................................................... 73
Conclusions............................................................................................................. 82
ii
CHAPTER 3: A simple Pichia pastoris production of an scFv-h3D6 and in vitro and in
vivo validation of its therapeutic effect. .................................................................... 83
Aims ........................................................................................................................ 85
Materials and Methods ......................................................................................... 86
Results and Discussion ........................................................................................... 92
Conclusions........................................................................................................... 103

CHAPTER 4: Effects of scFv-h3D6 on Aβ aggregation and astrocytic uptake are
modulated by apolipoprotein E and J mimetic peptides. ......................................... 105
Aims ...................................................................................................................... 107
Materials and Methods ....................................................................................... 108
Results .................................................................................................................. 111
Discussion ............................................................................................................. 120
CHAPTER 5: Effects on Aβ-induced inflammation of apolipoproteins E and J in
combination with scFv-h3D6. ................................................................................. 123
Aims ...................................................................................................................... 125
Materials and Methods ....................................................................................... 126
Results .................................................................................................................. 128
Discussion ............................................................................................................. 137
CHAPTER 6: In vivo study of the effect of scFv-h3D6 in combination with
apolipoprotein E or J mimetic peptides on Aβ pathology and inflammation ........... 141
Aims ...................................................................................................................... 143
Materials and Methods ....................................................................................... 144
Results .................................................................................................................. 147
Discussion ............................................................................................................. 155

V-GENERAL DISCUSSION ....................................................................................... 159

VI-CONCLUSIONS .................................................................................................. 167

VII-REFERENCES .................................................................................................... 173

iii
LIST OF TABLES AND FIGURES

FIGURES
I-INTRODUCTION ...................................................................................................... 1
Figure I-1 Extracellular Aβ plaques and intracellular neurofibrillary tangles (NFTs) ....... 3
Figure I-2. APP processing ............................................................................................... 4
Figure I-3. Aβ peptide generation and 3D-structure of its trimers ................................. 6
Figure I-4. ApoE3 NMR 3D-structure and isoforms .......................................................... 9
Figure I-5. Three-dimensional structure of some proteins related to Aβ clearance ..... 10
Figure I-6. ScFvs are a recombinant format in which the V and V domains of
H L
immunoglobulins are joined with a flexible polypeptide linker .................................... 16
Figure I-7. MTT toxicity assays with the SH-SY5Y human neuroblastoma cell-line ....... 19
Figure I-8. Secondary structure and thermal denaturation of scFv-h3D6 ..................... 20
Figure I-9. TEM of amyloid and WL fibrils by Aβ and scFv-h3D6 .................................... 21
Figure I-10. Immunoblotting analysis of the soluble Aβ-amyloid oligomers from
extracellular extracts of 5 mo-old NTg and 3xTg-AD mice and clusterin and apoE
concentrations in TBS extracts determined by ELISA ..................................................... 22
Figura I-11. Urea denaturation of ScFV-3D6h WT and its variants ................................ 23
Figure I-12. Representation of native and scrambled conformations of scFv-h3D6...... 24

III-GENERAL METHODS ........................................................................................... 29
Figure M-1. How Polymerase Chain Reaction works ..................................................... 31
Figure M-2. Circular Dichroism ....................................................................................... 35
Figure M-3. Thermal or chemical denaturation of a protein ......................................... 37
Figure M-4. An example of a DSC calorimetric scan ...................................................... 41
Figure M-5. Flow cytometry ........................................................................................... 45
Figure M-6. Schematic representation of the basic procedure performed in an ELISA. 46

iv
IV-CHAPTERS .......................................................................................................... 49

CHAPTER 1: Understanding the contribution of disulphide bridges to the folding and
misfolding of scFv-h3D6 .................................................................................................... 51
Figure 1.1. Thermal denaturation of V-DF followed by Trp-fluorescence and Circular
L
Dichroism (CD) ................................................................................................................ 55
Figure 1.2. Changes in the unfolding pathway and the aggregation tendency of the V -
H
DF mutant ....................................................................................................................... 57
Figure 1.3. Changes in the aggregation pathway of V -DF ............................................ 61
H
Figure 1.4. Effect of elongation mutations on V -DF ..................................................... 63
H
Figure 1.5. Diagrams for the folding and the aggregation pathways of scFv-h3D6 and
V -DF .............................................................................................................................. 64
H

CHAPTER 2: Towards the improvement in stability of scFv-h3D6 as a way to enhance its
therapeutic potential ....................................................................................................... 67
Figure 2.1. ScFv-h3D6 three-dimensional model and detail of the mutated sites ........ 73
Figure 2.2. Conformational study of the effects of K64R mutation alone or in
combination with the V elongation (C3) ....................................................................... 78
L
Figure 2.3. Analysis of the aggregation pathway ........................................................... 80

CHAPTER 3: A simple Pichia pastoris production of scFv-h3D6 and in vitro and in vivo
validation of its therapeutic effect. ................................................................................... 83
Figure 3.1. Mass spectometry MALDI-TOF and SDS-PAGE analysis of protein expression
at 48h, 72h, 96h and 120h after induction of EAEA and EAEV variants ......................... 94
Figure 3.2. ScFv-h3D6-Pp purification ............................................................................ 95
Figure 3.3. Large-scale fermentation profile ................................................................. 96
Figure 3.4. Protein Characterization .............................................................................. 97
Figure 3.5. Sequence of scFv-h3D6-Pp protein and disulfide bonds present in the scFv-
h3D6-Pp .......................................................................................................................... 99
Figure 3.6. Mass spectrometry analysis of scFv-h3D6-Pp and scFv-h3D6-Ec ............... 100
Figure 3.7. Therapeutic effects of scFv-hD6-Ec and scFv-h3D6-Pp .............................. 102

CHAPTER 4: Effects of scFv-h3D6 on Aβ aggregation and astrocytic uptake are modulated by
apolipoprotein E and J mimetic peptides. ........................................................................ 105
Figure 4.1. Secondary structure of the different molecules assayed ........................... 112
v