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`Michel Carney
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`Judicial-Proceedings-20190905-CARNEY
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`Title of thesis: Application of monoclonal antibodies to the investigation of the role of
`calcitonin gene-related peptide as a vasodilatory neurotransmitter
`Author:
`Keith Kwan Cheuk Tan.
`https://idiscover.lib.cam.ac.uk/permalink/f/t9gok8/44CAM_ALMA214296
`Permalink:
`48480003606
`Date:
`5 September 2019
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`

`

`Application of monoclonal antibodies to the investigation of
`the role of calcitonin gene-related peptide as a vasodilatory
`neurotransmitter
`
`Keith Kwan Cheuk Tan, BPharm, MSc, MRPharmS
`
`Gonville and Caius College, Cambridge
`
`A dissertation submitted to the University of Cambridge for the Ph.D. Degree
`
`

`

`Declaration
`
`TI1is dissertation is an account of my original work. However, a number of
`monoclonal antibodies were produced by others and made available to me as part of a
`research collaboration. These antibodies have been distinguished from those that I
`have produced, and their sources have been clearly stated. The characterization and
`application of these antibodies, reported in this dissertation, was entirely my own
`work.
`
`I hereby declare that this dissertation entitled "Application of monoclonal
`antibodies to the investigation of the role of calcitonin gene-related peptide as a
`vasodilatory neurotransmitter" is not substantially the same as any that I have
`submitted for a degree, diploma or other qualification at any other University.
`I further state that no part of my dissertation has already been or is being
`concurrently submitted for any such degree, diploma or other qualification.
`
`Date .............. ~/..t.'.!.~ .... .f.J .... ../}f 1:: ..... Signed ............. ,A.4(~ .......... ..
`
`,,,___.
`
`ii
`
`

`

`Acknowledgements
`
`The work described in this dissertation was performed in the Clinical Pharmacology
`Unit, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital,
`Cambridge and the Neuroscience Research Centre, Merck Sharp and Dohme Research
`Laboratories, Terlings Park, Harlow.
`I would like to thank Professor Morris Brown, Professor of Clinical
`Pharmacology and my supervisor, for his guidance and encouragement over the years;
`Dr. Shirley Ellis, Regional Pharmaceutical Adviser, East Anglian Regional Health
`Authority, for making it possible for me to embark on tbe PhD project; and Dr. Ray
`Hill, Director of Pharmacology, Merck Sharp and Dohme Research Laboratories, for
`his support during my work in the various laboratories under his management.
`This project would not have been completed without active interaction with
`some excellent scientists in Cambridge and Harlow. Many people have willingly
`taken time out of their own routines to teach me specialist skills, show me good
`practices, and warn me of the pitfalls. I am particularly grateful to Dr. Chris Plumpton
`for instruction on the techniques of monoclonal antibody production; Dr. Jenny
`Longmore for instruction on in viJro pharmacology techniques, Mr. David Smith and
`Dr. Mike Rigby for instruction on immunocytochemistry, Dr. Sara Shepheard and Ms.
`Debbie Cook for instruction on in vivo pharmacology techniques. The guidance and
`encouragement of Dr. Richard Hargreaves during my
`in vivo pharmacology
`experiments is gratefully acknowledged.
`This project was supported in part by a grant from the Locally Organized
`Research Scheme, East Anglian Regional Health Authority, and by a Hamett Fund
`scholarship awarded by the Faculty Board of Clinical Medicine, University of
`Cambridge.
`
`111
`
`

`

`Contents
`
`Chapter J General introduction
`
`Chapter 2
`
`Introduction to immunoblockade: pharmacokinetic and
`pharmacodynamic considerations
`
`Chapter 3 Development of monoclonal antibodies against CGRP
`
`Chapter 4 Characterization, purification and fragmentation of monoclonal antibodies
`against CGRP
`
`Chapter 5 Development of monoclonal antibodies against the CORP receptor
`
`Chapter 6 Characterization of CGRP receptor binding of monoclonal antibodies
`raised by an auto-anti-idiotypic approach or by immunization with
`purified CGRP receptor
`
`Chapter 7
`
`Phrumacological characterization of irnmunoblockade by an
`anti-CGRP monoclonal antibody
`
`Chapter 8 Demonstration of the neurotransmitter role of CORP by
`immunoblockade with monoclonal antibodies against CGRP
`
`Chapter 9
`
`In vivo jn,munoblookade studies with an unti-CORP monoclonal
`antibody and its Fab' fragment: role of CORP as an endogenous
`vasodi lator
`
`Chapter JO General discussion
`
`iv
`
`

`

`Contents
`
`Chapter l General introduction
`
`Chapter 2
`
`Introduction to immunoblockade: pharmacokinctic and
`pharmacodynamic considerations
`
`Chapter 3 Development of monoclonal antjbodies against CGRP
`
`Chapter 4 Characterization, purification and fragmentation of monoclonal antibodies
`against CGRP
`
`Chapter 5 Development of monoclonal antibodies against the CGRP receptor
`
`Chapter 6 Characterization of CGRP receptor binding of monoclonal antibodies
`raised by an auto-anti-idjotypic approach or by immunization with
`purified CGRP receptor
`
`Chapter 7
`
`Pharmacological characterization of immunoblockade by an
`anti-CGRP monoclonal antibody
`
`Chapter 8 Demonstration of the neurotransmitter role of CGRP by
`immunoblockade with monoclonal antibodies against CGRP
`
`Chapter 9
`
`In vivo immunoblockade studies with an anti-CORP monoclonal
`antibody and its Fab' fragment: role of CGRP as an endogenous
`vasodi lator
`
`Chapter 10 General discussion
`
`iv
`
`

`

`Correction ofp11ge numbers for Chapter I
`
`Chapter 1: General Introduction
`I. I. Calcitonin gene-related peptide ...................................... ........................................... I
`I. I.I. Structure of CORP ........................................... ., .............. .......................... 1
`1.1 .2. Distribution of CORP ................................................................................. 2
`1.1.3. 0istribulion of CORP binding sites ............................................................ 4
`J.1.4. Biological effects of CGRP ....................................................................... S
`1.1.5. CORP as a vasodilator ............................................................................... 6
`1.1.5.1. Mechanisms of vascular relaxation., .......................................... 8
`1.1.6. Effects of CORP on the heart ..................................................................... J 1
`I.I .7. Functional aspects of CORP receptors ....................................................... 12
`1.1.7.1. Receptor-effector coupllng ......................................................... 12
`1.1.7.2. Receptorantagonists ................................................................... 12
`1.1.7.3.Receplorsubtypes ...................................................................... 13
`1.1 .8. CORP as a neurotransm itter ....................................................................... 15
`I. 1.8.1. Criteria for a neurotransmitter .................................................... 15
`1.1.8.2. Capsaicin .................................................................................... 15
`I. 1.8.2.1. Release of CORP from capsaicin-sensitivc
`nerves .......................................................................................... 16
`1.1.8.2.2. Non-specific effects of capsaicin ................................ 16
`1.1.8.3. Metabolism of CORP ................................................................. 17
`1.1.9. Structurally-related peptides ....................................................................... 18
`1.l.9.1. Amylin ........................................................................................ 18
`1.1.9.2. Adrenomedullin ........................................................................ ~ 18
`I. 1.10. Physiological and pathophysiological roles ofCGRP .............................. 19
`I. I. JO. I . Neurogenic inJlaruruation ......................................................... 19
`1.l.10.2. Migraine .................................... , ............................................... 19
`(.1.10.3. Subarachnoid haemorrhage ...................................................... 20
`I .1.10.4. Raynaud's phenomenon ............................................................ 20
`I .J. I 0.5. Hypertension .................................................................. . ........ 20
`1.1.10.6. Pregnancy and fluid ovcrload ................................................... 21
`I. I. I 0. 7. Congestive cardiac failure ........................................................ 22
`1. 1,J0.8. Myocardial ischaemia ............................................................... 22
`1.l .10.9. Sepsis ........................................................................................ 22
`I .I . 10. 10. Other possible roles ................................................................ 23
`1.2. Monoclonal antibodies as pharmacological tools ....................................................... 23
`1.2. I. Antibodies ................................................................................................... 23
`1.2.2. Monoclonal antibodies ............................................................................... 24
`I .2.3. Anti-peptide MAbs: immunoblockade ....................................................... 24
`1.2.4. Anti-receptor MAhs: receptor antagonism ................................................. 25
`1.3. Aims of the project ..... - ............................................................................................. 26
`References ............................................................................................... 27
`
`Contents
`
`Chapter l: General Introduction
`I. I . Calcitonin gene-related peptide. ................................................................................. l
`I. I. I . Structure of CORP ...................................................................................... I
`1.1.2. Distribution ofCORP .................................................................................. 3
`1.1.3. Distribution of CORP binding sitcs .................................................. ., ....... .4
`1.1.4. Biological effects ofCGRP ......................................................................... 6
`I. I .5. CORP as a vasodilator ................................................................................ 7
`1. 1.5.1. Mechanisms of vascular relaxation ............................................. 9
`I . I .6. Effects or CGRP on the heart. ..................................................................... 12
`1.1 .7, Functional aspects of CORP receptors ........................................................ 13
`I .1.7.1. Receptor-effector coupling. ......................................................... 13
`1.1.7.2. Receptor antagonists ................................................................... 13
`1.1.7.3. Receptor subtypes ....................................................................... 14
`1.1 .8. CORP as a neurotransmitter ........................................................................ 16
`I. 1.8. I. Criteria for a ncurotr:msmitter ..................................................... 16
`1.1.8.2. Capsaicin ..................................................................................... 16
`1.1.8.2. I. Release of CORP from capsaicin-sensitive
`nerves .......................................................................................... 17
`I .1.8.2.2. Non-specific effects of capsaicin ................................ 18
`1.1.8.3. Metabolism ofCORP .................................................................. 18
`1.1.9. Structurally-related peptides ....................................................................... 19
`1.1.9.1. Amylin ........................................................................................ 19
`I .1.9,2. Adreuomedullin ......................................................................... .20
`I. 1.10. Physiological and pathophysiological roles of CORP .............................. 20
`I. I.I 0.1. Neurogenic inflammatiort ......................................................... 20
`l.l.10.2. Migraine ................................................................................... 21
`1.1.10.3. Subarachnoid haemorrhagc ....................................................... 21
`I .1 .10.4. Raynaud's phenomenon ............................................................ 22
`I. I. I 0.5. Hypertension ............................................................................. 22
`I.I, 10.6. Pregnancy and fluid overload ................................................... 23
`1. I .10.7. Congestive cardiac .failure ......................................................... 23
`I .1.10.8. Myocardial ischaemia ........................... ., .................................. 23
`1.1. I 0.9. Sepsis ........................................................................................ 24
`1.1.10.10. Other possible rok-s ................................................................. 24
`1.2. Monoclonal antibodies as pharmacological tools ....................................................... 25
`1.2. 1. Antibodi~s .................................................................. ., ............................... 2S
`1.2.2, Monoclonal antibodies .................................................................. ., ............ 26
`1.2.3. Anti-peptide MAbs: imnrnnoblockade ........................................................ 26
`1.2.4. Anti-receptor MAbs: receptor antagonism .................................................. 27
`1.3. Aim~ oftl1e project. ..................................................................................................... 28
`References ................................................................................................ 29
`
`Chapter 2: Jntroduction to immunoblockade: pharmacoldnctic anti
`pharmacodynamic considerations
`2.l. lntroduction ..................................................................................................... , ........... 54
`2.2. Phannacokinetics ........................................................................................................ 55
`2.2.I. Distribution ofantibodies.. .......................................................................... 55
`2.2.2. Elimination of antibodies ........ ................................................................... 57
`2.2.3. Pharmacokinetics of different antibody classcs .......................................... 58
`
`,,
`
`

`

`2.2.4. Pharmacokinetics of CORP ......................................................................... 58
`2.3. Pharmacodynamics ..................................................................................................... 60
`2.3. l. Mechanisms of immunoblockade ............................................................... 60
`2.3.2. Antibody-antigen interaction ....................................................................... 61
`2.3.3. Effect of antibody-ligand interaction on pharmacological rcsponse. .......... 62
`References ................................................................................................ 65
`
`Chapter 3: Development of monoclonal antibodies against CGRP
`3.1. lntroduetion ................................................................................................................. 69
`3.2. Methods ....................................................................................................................... 70
`3 .2. 1. Conjugation procedure ................................................................................ 70
`3.2.2. Immunization protocol ................................................................................ 70
`3.2.2.1. Preparation of antigen in Freund's adjuvanl ............................... 70
`3.2.2.2. lm1nunization schedule ............................................................... 7 J
`3.2.2.3. Screening of serum for anti-CGRP antibodics ............................ 71
`3.2.3. Enzyme-linked immunoadsorbent assay (ELTSA) ...................................... 7 1
`3.2.3. 1. Development of indirect ELISA screening assay ....................... 72
`3.2.3.2. Experimental procedures ............................................................. 72
`3.2.4. Radioimmunoassay (RIA) ........................................................................... 73
`3.2.S. Preparation of feeder layer cells .................................................................. 74
`3.2.6. Preparation ofmyeloma cells ...................................................................... 74
`3.2.7. Fusion procedure ......................................................................................... 74
`3.2.7. 1. Experimental procedures ............................................................. 75
`3 .2.8. Post-fusion management ........................... .................................................. 77
`3.2.9. Screening of supernatants ........................................................................... 77
`3 .2.10. Selection of positive hybridoma cells for cloning. .................................... 77
`3.2.1 l. Cloning by limiting dilution ...................................................................... 78
`3 .2.12. Cryopreservation of hybridoma cells ........................................................ 78
`3 .2. 13. Thawing of cryopreserved cells ................................................................ 78
`3.2.14. Cryopreservation of spleen cells ............................................................... 79
`3 .2. l S. Bulk production of MAbs in vivo .............................................................. 79
`3.2. 16. Bulk production of MAbs in vilro ............................................................. 80
`3.3. Results .... , .................................................................................................................... 80
`3.3.1. lmmunizations ............................................................................................. 80
`3.3 .2. Fusions ..................................... ................................................................... 81
`3.3.3. CloniJ1g of selected cell lines ...................................................................... 81
`3.4. Discussion ................................................................................................................. ,.86
`References ............................ .................................................................... 88
`
`Chapter 4: Characterization, purification and fragmentation of monoclonal
`antibodies against CGRP
`4.1. lntroduction ................................................................................................................. 90
`4.2. MetJiods ....................................................................................................................... 91
`4.2. 1. ELISA, receptor binding assay and RIA. .................................................... 91
`4.2.2. Determination of antibody class. ................................................................. 92
`4.2.3. Determination of protein concentration ...................................................... 92
`4 .2.4. Purification ofMAbs ..................................................... .............................. 93
`4.2.4. I. Ammonium sulphate precipitation .............................................. 93
`4.2.4. 1. 1. Principlos ..................................................................... 93
`4.2.4. 1.2. Preparation of saturated ammonium sulphate
`solution ........................................................................................ 94
`4.2.4.l.3. Experimental procedures: ascites fluid ....................... 94
`
`vi
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`

`

`4.2.4. l .4. Experimental procedures: hybridoma culture
`supernatant .................................................................................. 94
`4.2.4.2. Protein A sepharose affinity chromatography ............................. 95
`4.2.5. Fragmentation of MAbs .............................................................................. 95
`4.2.5.1 . Preparation of F(ab'h by pepsin digestion ................................. 95
`4 .2.5. I. I . Experimental procedures ............................................. 96
`4.2.5.2. Preparation of Fab' fragments from F(ab')i fragments ............... 96
`4.2.5.2. 1. Experimental procedures ............................................. 97
`4.2.5.3. Concentration of F(ab')i and Fab' fragments .............................. 97
`4.2.5.4. Indirect ELISA of F(ab'h and Fab' fragments ............................ 97
`4.2.6. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis ................... 98
`4.2.6. 1. Analysis of antibody fragmentation by SOS-PAGE ................... I 00
`4.2.7. lmtnunocytochemistry ................................................................................. 10 1
`4.3. Results ......................................................................................................................... 102
`4.3.1. ELISA, receptor binding assay and RJA .......... ........................................... 102
`4.3.2. Determination of antibody class. ................................................................. 103
`4.3.J. Purification and fragmentation ofMAbs .................................................... .1 03
`4.3.4. Tmmunocytochemistry ................................................................................. 104
`4.4 Discussion .................................................................................................................... 115
`References ................................................................................................ 11 7
`
`Chapter 5: Development of monoclonal antibodies against the CGRP receptor
`5. J. Introduction ................................................................................................................. l 19
`5.2. Methods ....................................................................................................................... 119
`5.2. 1. Preparation of rat liver membranes ............................................................. 120
`5.2.2. Preparation of bovine cerebellum membranes ............................................ 120
`5.2.3. Determination of protein concentration of membrane preparations ........... 120
`5.2.4. Receptor binding assay of CORP using rat liver membrane
`preparation ............................................................................................................ 120
`5.2.4.1. Reduction of non-specific binding by siliconization and
`use of BSA ............................................................................................... 121
`5.2.4.2. Estimation ofreceptor binding parameters ................................. 121
`5.2.5. Use of receptor binding assay for screening scrum and cell culture
`supen1atants ........................................................................................................... 122
`5.2.6. Receptor binding assay of CORP using bovine cerebellum
`membrane preparation ........................................................................................... 122
`5.2.7. Dot immunobinding assay for immunoglobulin in supernatants ................ 122
`5.2.8. In vivo immunization protocol and screening for anti-receptor
`antibodies .............................................................................................................. 123
`5.2.9. Jn vitro immunization protocol and fusion. ................. ................................ 124
`5.3. Results ......................................................................................................................... 125
`5.3.1. Receptor binding assay ............................................................................... 125
`5 .3 .2. In vivo immunization ................................................................................... 126
`5.3.3. In vitro immunization .................................................................................. l 26
`5.4. Discussion ................................................................................................................... l27
`References ................................................................................................ t 30
`
`Chapter 6: Characterization of CG RP receptor binding of monoclonal
`antibodies raised by an auto-anti-idiotypic approach or by
`immunization with purified CGRP receptor
`6. 'I. Introduction ................................................................................................................. 132
`6.2. Methods ............................................... ........................................................................ 134
`
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`6.2.1. Source of potential anti-receptor MAbs studicd .......................................... 134
`6.2.2. Receptor binding studies ............................................................................. 134
`Materials ................... , .................................................................. 134
`6.2.2. l . Experimental procedures ............................................................. 135
`6.2.2. 1.1. Rat liver membrane preparation .................................. 135
`6.2.2.1.2. Rat whole brain membrane preparation ...................... 135
`6.2.2.1.3. SK-N-MC human neuroblastoma cell membrane
`preparation .................................................................................. 135
`6.2.2.1.4. Binding assay (rat whole brain or SK-N-MC cell
`membrane preparation) ............................................................... 136
`6.2.3. lmmunocytochemistry ................................................................................. 136
`6.2.3.1. Principles ..................................................................................... 136
`6.2.3.2. Transcardiac perfusion fixation. .................................................. 138
`6.2.3.2. 1. Experimental procedurcs ............................................. 139
`6.2.3.3. Snap freezing of tissucs ........................................ ....................... 139
`6.2.3.4. Cryostat sections ......................................................................... 139
`6.2.3.5. lmmunocytochemical staining of free-floating tissue
`sections ..................................................................................................... 140
`6.2.3.5.1. Optimization of staining procedure ............................. t 40
`6.2.3.5.2. Experimental procedures ............................................. 140
`6.2.3.5.3. Experimental controls ................................................. 141
`6.2.3.6. lmmunocytochcmistry using fresh (unfixed) tissue
`sections ......................................... ............................................................ 141
`6.2.3. 7. Immunocytochemistry of cultured cells ...................................... 141
`6.2.3.7.1. Cell culturc .................................................................. 142
`6.2.3.7.2. Coating ofcoverslips with poly-L-lysine. ................... 142
`6.2.3.7.3 Experimental procedures .............................................. 143
`6.2.3.8. Microscopy and Photography ..................................................... 143
`6.2.3 .9. lmage Analysis ............................................................................ 144
`6.2.4. Receptor autoradiography .. ......................................................................... 144
`6.2.4.1. Principles ..................................................................................... l 44
`6.2.4.2. Experimental procedures ............................................................. 145
`6.2.5. Enzyme-linked immunoadsorbcnt assay (ELISA) ...................................... 145
`6.2.5. 1. ELISAs to investigate the 11intemal image" property of Id
`MAbs ........................................................................................................ 146
`6.2.5.2. ELISAs to investigate the potential anti-immunoglobulin
`binding ofld MAbs .................................................................................. 146
`6.3. Results ......................................................................................................................... 147
`6.3.1. Auto-anti-idiotypic approach: Id MAbs ...................................................... 147
`6.3.1. 1. Receptor binding studies ............................................................. 147
`6.3. 1.2. lmmunoeytochemistry and receptor autoradiography. ................ 147
`6.3. 1.3. ELISAs ........................................................................................ 148
`6.3.2. Anti-receptor MAbs: RCG MAbs ............................................................... 148
`6.4. Discussion .................... ............................................................................................... l57
`References ................................................................................................ 161
`
`Chapter 7: Pharmacological characterization of immunoblockade by an anti(cid:173)
`CGRP monoclonal antibody
`7.1. Introduction ................................................................................................................. 166
`7.2. Methods ....................................................................................................................... 167
`7.2. 1. Experimental procedures ............................................................................. 168
`7.2.2. Blockade of responses to exogenous CGRP ............................................... 169
`
`viii
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`

`

`7.2.3. Blockade of responses to capsaicin ............................................................. 170
`7.2.4. Data analysis ............................................................................................... 170
`7.3. Results ......................................................................................................................... 171
`7.3.1 . Blockade of responses to exogenous CGRP ............................................... 17 l
`7.3.2. Blockade of responses to capsaicin ............................................................. 171
`7.4. Discussion ................................................................................................................... 174
`Refcrences ................................................................................................ 176
`
`Chapter 8: Demonstration of the neurotransmitter role of CGRP by
`immunoblockade with monoclonal antibodies against CGRP
`8.1 . Introduction ................................................................................................................. 179
`8.2. Methods ....................................................................................................................... 179
`8.2.1. Tissue bath experiments .............................................................................. 180
`8.2.2. Modelling of immunoblockade ................................................................... 182
`8.2.3. Statistical analysis ......................... .............................................................. 182
`8.3. Results ......................................................................................................................... 184
`8.3.1. lmmunoblockade of exogenous CGRP ....................................................... 184
`8.3.2. Modelling of immunoblockadc ................................................................... 185
`8.3.3. Immunoblockade of endogenous CGRP ..................................................... J 94
`8.4. Discussion ...............................................................................................