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