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
`
`Lilly Exhibit 1287
`Eli Lilly & Co. v. Teva
`Pharms. Intl GMBH
`
`

`

`Declaration
`
`This 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.
`
`6/ 7
`
`Date
`
`Signed
`
`11
`
`

`

`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.
`to thank Professor Morris Brown, Professor of Clinical
`I would
`like
`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 the 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 vitro 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 Harnett Fund
`scholarship awarded by the Faculty Board of Clinical Medicine, University of
`Cambridge.
`
`111
`
`

`

`Contents
`
`Chapter 1 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 CGRP 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
`
`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 -CGRP monoclonal
`antibody and its Fab' fragment: role of CGRP as an endogenous
`vasodilator
`
`Chapter 10 General discussion
`
`iv
`
`

`

`Contents
`
`Chapter 1
`
`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 CGRP 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
`
`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 -CGRP monoclonal
`antibody and its Fab' fragment: role of CGRP as an endogenous
`vasodilator
`
`Chapter 10 General discussion
`
`iv
`
`

`

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`Chapter 2: Introduction to immunoblockade: pharmacokinetic and
`
`pharmacodynamie considerations
`
`2.2.3. Pharmacokinetics of different antibody classes
`2.2.2. Elimination of antibodies
`2.2.1. Distribution of antibodies.
`
`2.2. Pharmacokinetics
`2.1. Introduction
`
`1.1.10. Physiological and pathophysiological roles of CGRP
`
`1.1.8.2.2. Non -specific effects of capsaicin
`nerves
`1.1.8.2.1. Release of CGRP from capsaicin- sensitive
`
`1.1.8.3. Metabolism of CGRP
`
`1.1.9. Structurally- related peptides
`
`1.1.9.2. Adrenomedullin
`1.1.9.1. Amylin
`
`1.1.5.1. Mechanisms of vascular relaxation
`
`1.1.8. CGRP as a neurotransmitter
`1.1.7.3. Receptor subtypes
`1.1.72. Receptor antagonists
`1.1.7.1. Receptor- effector coupling.
`I .1.7. Functional aspects of CGRP receptors
`1.1.6. Effects of CGRP on the heart
`
`1.1.8.2. Capsaicin
`1.1.8.1. Criteria for a neurotransmitter
`
`1.1.5. CGRP as a vasodilator
`1.1.4. Biological effects of CGRP
`1.1.3. Distribution of CGRP binding sites
`1.1.2. Distribution of CGRP
`1.1.1. Structure of CGRP
`
`1.1. Calcitonin gene -related peptide.
`Chapter 1: General Introduction
`
`Contents
`
`1.2.4. Anti -receptor MAbs: receptor antagonism.
`1.2.3. Anti -peptide MAbs: immunoblockade
`1.2.2. Monoclonal antibodies
`1.2.1. Antibodies
`
`References
`
`1.3. Aims of the project
`
`1.2. Monoclonal antibodies as pharmacological tools
`
`1.1.10.10. Other possible roles
`1.1.10.9. Sepsis
`1.1.10.8. Myocardial ischaemia
`1.1.10.7. Congestive cardiac failure
`1.1.10.6. Pregnancy and fluid overload
`1.1.10.5. Hypertension
`1.1.10.4. Raynaud's phenomenon
`1.1.I0.3. Subarachnoid haemorrhage
`1.1.10.2. Migraine
`1.1.10.1. Neurogenic inflammation.
`
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`2
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`1
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`1
`
`1.1.10. Physiological and pathophysiological roles of CGRP
`
`1.1.8.2.2. Non -specific effects of capsaicin
`nerves
`1.1.8.2.1. Release of CGRP from capsaicin -sensitive
`
`1.1.9. Structurally -related peptides
`
`1.1.9.2. Adrenomedullin
`1.1.9.1. Amylin
`
`1.1.8.3. Metabolism of CGRP
`
`1.1.8.2. Capsaicin
`1.1.8.1. Criteria for a neurotransmitter
`
`1.1.5.1. Mechanisms of vascular relaxation
`
`1.1.8. CGRP as a neurotransmitter
`1.1.7.3. Receptor subtypes
`1.1.7.2. Receptor antagonists
`1.1.7.1. Receptor -effector coupling
`1.1.7. Functional aspects of CGRP receptors
`1.1.6. Effects of CGRP on the heart
`
`1.1.5. CGRP as a vasodilator
`1.1.4. Biological effects of CGRP
`1.1.3. Distribution of CGRP binding sites
`1.1.2. Distribution of CGRP
`1.1.1. Structure of CGRP
`
`1.1. Calcitonin gene -related peptide
`Chapter 1: General Introduction
`
`Correction of page numbers for Chapter 1
`
`1.2.4. Anti -receptor MAbs: receptor antagonism
`1.2.3. Anti- peptide MAbs: immunoblockade
`1.2.2. Monoclonal antibodies
`1.2.1. Antibodies
`
`References
`
`1.3. Aims of the project
`
`1.2. Monoclonal antibodies as pharmacological tools
`
`1.1.10.10. Other possible roles
`1.1.10.9. Sepsis
`1.1.10.8. Myocardial ischaemia
`1.1.10.7. Congestive cardiac failure
`1.1.10.6. Pregnancy and fluid overload
`1.1.10.5. Hypertension
`1.1.10.4. Raynaud's phenomenon
`1.1.10.3. Subarachnoid haemorrhage
`1.1.10.2. Migraine
`1.1.10.1. Neurogenic inflammation
`
`_
`_
`-
`_
`
`_
`
`-
`
`

`

`2.2.4. Pharmacokinetics of CGRP
`2.3. Pharmacodynamics
`2.3.1. Mechanisms of immunoblockade
`2.3.2. Antibody- antigen interaction
`2.3.3. Effect of antibody -ligand interaction on pharmacological response
`References
`
`Chapter 3: Development of monoclonal antibodies against CGRP
`3.1. Introduction
`3.2. Methods
`3.2.1. Conjugation procedure
`3.2.2. Immunization protocol
`3.2.2.1. Preparation of antigen in Freund's adjuvant
`3.2.2.2. Immunization schedule
`3.2.2.3. Screening of serum for anti -CGRP antibodies
`3.2.3. Enzyme- linked immunoadsorbent assay (ELISA)
`3.2.3.1. Development of indirect ELISA screening assay
`3.2.3.2. Experimental procedures
`3.2.4. Radioimmunoassay (RIA)
`3.2.5. Preparation of feeder layer cells
`3.2.6. Preparation of myeloma cells
`3.2.7. Fusion procedure
`3.2.7.1. Experimental procedures
`3.2.8. Post -fusion management
`3.2.9. Screening of supernatants
`3.2.10. Selection of positive hybridoma cells for cloning
`3.2.11. Cloning by limiting dilution
`3.2.12. Cryopreservation of hybridoma cells
`3.2.13. Thawing of cryopreserved cells
`3.2.14. Cryopreservation of spleen cells
`3.2.15. Bulk production of MAbs in vivo
`3.2.16. Bulk production of MAbs in vitro
`3.3. Results
`3.3.1. Immunizations
`3.3.2. Fusions
`3.3.3. Cloning of selected cell lines
`3.4. Discussion
`
`References
`
`58
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`60
`61
`62
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`88
`
`Chapter 4: Characterization, purification and fragmentation of monoclonal
`antibodies against CGRP
`4.1. Introduction
`4.2. Methods
`4.2.1. ELISA, receptor binding assay and RIA
`4.2.2. Determination of antibody class
`4.2.3. Determination of protein concentration
`4.2.4. Purification of MAbs
`4.2.4.1. Ammonium sulphate precipitation
`4.2.4.1.1. Principles
`4.2.4.1.2. Preparation of saturated ammonium sulphate
`solution
`4.2.4.1.3. Experimental procedures: ascites fluid
`
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`
`vi
`
`

`

`4.2.4.1.4. Experimental procedures: hybridoma culture
`supernatant
`4.2.4.2. Protein A sepharose affinity chromatography
`4.2.5. Fragmentation of MAbs
`4.2.5.1. Preparation of F(ab')2 by pepsin digestion
`4.2.5.1.1. Experimental procedures
`4.2.5.2. Preparation of Fab' fragments from F(ab')2 fragments
`4.2.5.2.1. Experimental procedures
`4.2.5.3. Concentration of F(ab')2 and Fab' fragments
`4.2.5.4. Indirect ELISA of F(ab1)2 and Fab' fragments
`4.2.6. Sodium dodecyl sulphate -polyacrylamide gel electrophoresis
`4.2.6.1. Analysis of antibody fragmentation by SDS -PAGE
`4.2.7. Immunocytochemistry
`4.3. Results
`4.3.1. ELISA, receptor binding assay and RIA
`4.3.2. Determination of antibody class
`4.3.3. Purification and fragmentation of MAbs
`4.3.4. Immunocytochemistry
`4.4 Discussion
`
`References
`
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`101
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`104
`115
`117
`
`Chapter 5: Development of monoclonal antibodies against the CGRP receptor
`119
`5.1. Introduction
`119
`5.2. Methods
`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 CGRP using rat liver membrane
`preparation
`5.2.4.1. Reduction of non -specific binding by siliconization and
`use of BSA
`5.2.4.2. Estimation of receptor binding parameters
`5.2.5. Use of receptor binding assay for screening serum and cell culture
`supernatants
`5.2.6. Receptor binding assay of CGRP using bovine cerebellum
`membrane preparation
`5.2.7. Dot immunobinding assay for immunoglobulin in supernatants
`5.2.8. In vivo immunization protocol and screening for anti- receptor
`antibodies
`5.2.9. In vitro immunization protocol and fusion
`5.3. Results
`5.3.1. Receptor binding assay
`5.3.2. In vivo immunization
`5.3.3. In vitro immunization
`5.4. Discussion
`
`References
`
`Chapter 6: Characterization of CGRP receptor binding of monoclonal
`antibodies raised by an auto -anti -idiotypic approach or by
`immunization with purified CGRP receptor
`6.1. Introduction
`6.2. Methods
`
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`
`120
`
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`121
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`122
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`

`6.2.1. Source of potential anti -receptor MAbs studied
`6.2.2. Receptor binding studies
`Materials
`6.2.2.1. Experimental procedures
`6.2.2.1.1. Rat liver membrane preparation
`6.2.2.1.2. Rat whole brain membrane preparation
`6.2.2.1.3. SK -N -MC human neuroblastoma cell membrane
`preparation
`6.2.2.1.4. Binding assay (rat whole brain or SK -N -MC cell
`membrane preparation)
`6.2.3. Immunocytochemistry
`6.2.3.1. Principles
`6.2.3.2. Transcardiac perfusion fixation.
`6.2.3.2.1. Experimental procedures
`6.2.3.3. Snap freezing of tissues
`6.2.3.4. Cryostat sections
`6.2.3.5. Immunocytochemical staining of free -floating tissue
`sections
`6.2.3.5.1. Optimization of staining procedure
`6.2.3.5.2. Experimental procedures
`6.2.3.5.3. Experimental controls
`6.2.3.6. Immunocytochemistry using fresh (unfixed) tissue
`sections
`6.2.3.7. Immunocytochemistry of cultured cells
`6.2.3.7.1. Cell culture
`6.2.3.7.2. Coating of coverslips with poly -L- lysine
`6.2.3.7.3 Experimental procedures
`6.2.3.8. Microscopy and Photography
`6.2.3.9. Image Analysis
`6.2.4. Receptor autoradiography
`6.2.4.1. Principles
`6.2.4.2. Experimental procedures
`6.2.5. Enzyme -linked immunoadsorbent assay (ELISA)
`6.2.5.1. ELISAs to investigate the "internal image" property of Id
`MAbs
`6.2.5.2. ELISAs to investigate the potential anti -immunoglobulin
`binding of Id MAbs
`
`6.3. Results
`6.3.1. Auto -anti -idiotypic approach: Id MAbs
`6.3.1.1. Receptor binding studies
`6.3.1.2. Immunocytochemistry and receptor autoradiography
`6.3.1.3. ELISAs
`6.3.2. Anti -receptor MAbs: RCG MAbs
`6.4. Discussion
`
`References
`
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`161
`
`Chapter 7: Pharmacological characterization of immunoblockade by an anti -
`CGRP monoclonal antibody
`7.1. Introduction
`7.2. Methods
`7.2.1. Experimental procedures
`7.2.2. Blockade of responses to exogenous CGRP
`
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`viii
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`

`7.2.3. Blockade of responses to capsaicin
`7.2.4. Data analysis
`7.3. Results
`7.3.1. Blockade of responses to exogenous CGRP
`7.3.2. Blockade of responses to capsaicin
`7.4. Discussion
`
`References
`
`Chapter 8: Demonstration of the neurotransmitter role of CGRP by
`immunoblockade with monoclonal antibodies against CGRP
`8.1. Introduction
`8.2. Methods
`8.2.1. Tissue bath experiments
`8.2.2. Modelling of immunoblockade
`8.2.3. Statistical analysis
`8.3. Results
`8.3.1. Immunoblockade of exogenous CGRP
`8.3.2. Modelling of immunoblockade
`8.3.3. Immunoblockade of endogenous CGRP
`8.4. Discussion
`
`References
`
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`
`Chapter 9: In vivo immunoblockade studies with an anti -CGRP monoclonal
`antibody and its Fab' fragment: role ofCGRP as an endogenous
`vasodilator
`9.1. Introduction
`9.2. Methods
`9.2.1. Effect of exogenous RaCGRP on blood pressure
`9.2.1.1. Experimental procedures
`9.2.1.2. Blockade of blood pressure response with MAb C4.19 IgG
`9.2.1.3. Blockade of blood pressure response with MAb C4.19
`Fab' fragment
`9.2.1.4. Blockade of blood pressure response with HaCGRP8_37
`9.2.2. Change in skin blood flow measured by Laser Doppler flowmetry
`following antidromic stimulation of the saphenous nerve
`9.2.2.1. Principles
`9.2.2.2. Experimental procedures
`9.2.2.3. Quantification
`9.2.2.4. Reproducibility study
`9.2.2.5. Determination of sample size
`9.2.2.6. Effect of MAb C4.19 IgG
`9.2.2.7. Effect of MAb C4.19 Fab' fragment
`9.2.2.8. Effect of normal mouse Fab' fragment
`9.2.2.9. Effect of HaCGRP8_37
`9.2.2.10 Effect of co- administration of HaCGRP8_37 and RP-
`67,580
`9.2.3. Effect of MAb C4.19 Fab' fragment on the pressor response to
`angiotensin II
`9.2.4. Data analysis
`9.3. Results
`9.3.1 Effects on baseline MAP
`9.3.2 Blockade of the effect of RaCGRP on MAP
`
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`

`9.3.2.1. Blockade of MAP response with MAb C4.19 IgG
`9.3.2.2. Blockade of MAP response with MAb C4.19 Fab'
`fragment
`9.3.2.3. Blockade of MAP response with HaCGRP8_37
`9.33 Change in skin blood flow by measured by Laser Doppler flowmetry
`following antidromic stimulation of the saphenous nerve
`9.3.3.1. Reproducibility study
`9.3.3.2. Effect of MAb C4.19 IgG
`9.3.3.3. Effect of MAb C4.19 Fab' fragment
`9.3.3.4. Effect of normal mouse Fab' fragment
`9.3.3.5. Comparison of the effects of MAb C4.19 Fab' fragment
`and normal mouse Fab' fragment
`9.3.3.6. Effect of HaCGRP8_37
`9.3.3.7. Comparison of the effects of MAb C4.19 Fab' fragment
`and HaCGRP8_37
`9.3.3.8. Co- administration of HaCGRP8_37 and RP- 67,580
`9.3.4 Effect of MAb C4.19 Fab' fragment on the pressor response to
`angiotensin II
`9.4. Discussion
`9.4.1. Immunoblockade of the MAP response to exogenous RaCGRP
`9.4.2. Increase in skin blood flow following antidromic stimulation of the
`saphenous nerve: immunoblockade of endogenous CGRP
`9.4.3. Comparison of CGRP blockade by Fab' fragment or HaCGRP8 -37
`9.4.4. Co- administration of HaCGRP8 -37 and RP- 67,580
`9.4.5. Effect of MAb C4.19 Fab' fragment on the pressor response to
`angiotensin II
`9.4.6. Effect of MAb C4.19 IgG and Fab' fragment on baseline MAP
`9.4.7. Application of MAb Fab' fragments in immunoblockade studies
`References
`
`Chapter 10: General discussion
`10.1. Critique of immunoblockade
`10.1.1. In vitro evaluation of antibodies
`10.1.2. Positive control experiments
`10.1.3. Specificity controls
`10.1.4. Use of monoclonal antibodies and Fab' fragments
`10.2. Immunoblockade of CGRP
`10.3. Comparison of immunoblockade and receptor antagonism
`10.4. Critique of anti -receptor monoclonal antibodies.
`10.4.1. Production of monoclonal antibodies against receptors
`10.4.2. Pharmacodynamic properties of anti -receptor antibodies
`10.5. Future directions
`References
`
`209
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`215
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`

`

`AUC
`
`Bis
`BSA
`
`Bmax
`CDR
`CGRP
`cpm
`95% C.I.
`DAB
`DMEM
`EC50
`EDTA
`ELISA
`
`Emax
`2FD, 10FD, 20FD
`
`FITC
`Fmax
`
`HAT
`HaCGRP
`HaCGRP8_37
`HaCGRP
`HT
`i.p.
`
`i.v.
`
`KC1
`Kd
`MAb
`MAP
`NK 1
`PAbs
`PBS
`PBSTx
`
`Abbreviations
`
`Area under the flux -time curve attributable to nerve
`stimulation
`N'N'- Bis -methylene -acrylamide
`Bovine serum albumin
`Concentration of binding sites
`Complementarity -determining region
`Calcitonin gene -related peptide
`Counts per minute
`95% Confidence interval
`3,3' Diaminobenzidine
`Dulbecco's modified Eagles medium
`Concentration which produces half -maximal effect
`Ethylene- diamine -tetraacetic acid
`Enzyme -linked immunoadsorbent assay
`Maximum effect
`Dulbecco's modified Eagles medium containing 2, 10,
`20% foetal calf serum
`Fluorescein isothiocyanate
`Maximum change in skin blood flow attributable to nerve
`stimulation
`Hypoxanthine, aminopterin and thymidine
`Human a CGRP
`C- terminal (8 -37) fragment of HaCGRP
`Human ß CGRP
`Hypoxanthine and thymidine
`Intraperitoneal
`Intravenous
`Potassium chloride
`Dissociation constant
`Monoclonal antibody
`Mean arterial pressure
`Neurokinin 1
`Polyclonal antibodies
`Phosphate -buffered saline
`0.1M PBS /0.3% Triton -X 100
`
`xi
`
`

`

`PEG
`PMSF
`RaCGRP
`RaCGRP
`RIA
`rpm
`SDS -PAGE
`
`SFD
`SHR
`TEMED
`Tris
`TSH
`
`Polyethylene glycol
`Phenylmethyl- sulphonyl- fluoride
`Rata CGRP
`Rat ß CGRP
`Radioimmunoassay
`Revolutions per minute
`Sodium dodecyl sulphate -polyacrylamide gel
`electrophoresis
`Serum -free Dulbecco's modified Eagles medium
`Spontaneously hypertensive rat
`N, N, N ',N- tetramethylethylenediamine
`Tris(hydroxymethyl)aminomethane
`Thyroid -stimulating hormone
`
`)di
`
`

`

`Summary
`
`Calcitonin gene- related peptide (CGRP) is produced by alternative rRNA processing
`of the calcitonin gene. It is a potent vasodilator and is localized in perivascular
`sensory neurons. The localization of CGRP -immunoreactivity in primary afferent
`neurons innervating many different tissues and the wide distribution of CGRP binding
`sites suggest that CGRP may be a physiologically important neurotransmitter. The
`aim of the project was to investigate whether vasodilatory responses to CGRP
`released from perivascular sensory nerves could be blocked with anti -CGRP or anti -
`CGRP receptor monoclonal antibodies (MAbs).
`MAbs against CGRP were successfully produced and characterized for their
`ability to inhibit CGRP receptor binding (immunoblockade). Unsuccessful attempts
`were made to develop MAbs against the CGRP receptor by in vivo and in vitro
`immunization of animals with CGRP receptor -rich membranes and by an auto -anti-
`idiotypic approach.
`Eleven MAbs against CGRP were screened for immunoblocking properties in
`an isolated porcine coronary artery assay or an electrically -stimulated isolated rat vas
`deferens assay. MAb C4.19 was identified as a MAb that effectively blocks the
`effects of exogenous rat CGRP. It was demonstrated that the pharmacological
`response to CGRP in the presence of MAb C4.19 could be predicted when the
`dissociation constant and concentration of binding sites of the antibody were known.
`Capsaicin was used to stimulate the release of endogenous CGRP from primary
`afferent neurons. Capsaicin- induced inhibition of electrically- stimulated contractions
`of the isolated rat vas deferens was significantly attenuated by MAb C4.19. The
`results of the in vitro immunoblockade studies suggest that CGRP has a major role as
`a neurotransmitter at the neuroeffector junction of the rat vas deferens.
`the hypotensive response to exogenous
`The effect of MAb C4.19 on
`rat aCGRP (RaCGRP) was investigated in the pentobarbitone- anaesthetized rat. The
`role of CGRP in mediating antidromic vasodilatation was investigated by blockade of
`the increase in hind paw skin blood flow produced by saphenous nerve stimulation in
`the pentobarbitone- anaesthetized rat. Change in skin blood flow was measured by
`laser Doppler flowmetry. The dose -response relationship for the effect of i.v.
`RaCGRP was similarly shifted rightward by MAb C4.19 IgG (1 mg /rat i.v.) and Fab'
`fragment (2 mg /rat i.v.). The C- terminal fragment of human aCGRP (HaCGRP8_37;
`100 nmol /kg i.v.) also blocked the hypotensive effect of RaCGRP significantly. MAb
`C4.19 Fab' fragment (2 mg /rat i.v.) and HaCGRP8_37 (100 nmol /kg i.v.) but not
`MAb C4.19 IgG (up to 3 mg /rat i.v.) blocked the increased skin blood flow response
`to antidromic stimulation of the saphenous nerve. Normal mouse Fab' fragment
`
`s
`
`

`

`(2 mg /rat i.v.) had no significant effect on antidromic skin vasodilatation. The mean
`percentage changes in skin blood flow parameters due to MAb C4.19 Fab' fragment
`were significantly different from those due to normal mouse Fab' fragment but not
`from those due to HaCGRP8_37.
`The results of this project show that immunoblockade with an anti -CGRP
`MAb may be used to demonstrate the physiological role of endogenous CGRP.
`However, Fab' fragments should be used for acute in vivo pharmacological studies to
`ensure effective distribution to the site of action. The results of immunoblockade
`agree with those obtained by receptor blockade with HaCGRP8_37 and provide
`in support of the role of CGRP in mediating skin
`complementary evidence
`vasodilatation.
`
`xiv
`
`

`

`Perhaps more significantly, there are major structural similarities between
`
`(Goltzman & Mitchell, 1985; Wimalawansa & El- Kholy, 1993).
`Salmon calcitonin at high concentrations can cross -react with CGRP receptors
`levels and inhibiting osteoclastic activity (Zaidi et al., 1988; Raue et al., 1987).
`about 100- to 1000 -fold less potent than human calcitonin in lowering plasma calcium
`The different forms of CGRP cross -react weakly with calcitonin receptors and are
`homology between human aCGRP (HaCGRP) and human calcitonin is only 16 %.
`CGRP shares limited structural similarities with calcitonin. The sequence
`
`from the spinal cord of the pig (Kimura et al., 1987).
`CGRP with striking homology to the rat and human CGRPs has also been isolated
`differ in three amino acid positions in man but only one in the rat (Figure 1.1). A
`linked by a disulphide bridge and an amidated C- terminus. The two forms of CORP
`CGRP consists of 37 amino acids with an N- terminal 6 -amino acid ring structure
`
`1.1.1. Structure of CGRP
`
`chromosome 11.
`second calcitonin. The calcitonin/aCGRP and 13CORP genes are both located on
`processed to form multiple mRNAs. Thus the CGRP gene does not code for a
`distinct 3' terminal exons. The transcripts from the (CGRP gene, however, are not
`aCGRP. The fully processed mRNAs have the first 3 exons in common but contain
`containing 6 exons which are subsequently spliced to form either calcitonin or
`The calcitonin/aCGRP gene is transcribed as a larger precursor mRNA
`
`CGRP, now referred to as aCGRP.
`The second peptide is termed CGRP to distinguish it from the originally described
`subsequently identified in rat and man (Amara et aL, 1985; Steenbergh et al., 1985).
`A second gene encoding a closely homologous 37 -amino acid peptide was
`
`medullary thyroid carcinoma tissue (Morris et al., 1984).
`the production of calcitonin or CGRP. Human CGRP was first isolated from
`Alternative processing of the primary mRNA transcript of the calcitonin gene leads to
`studies of the rat calcitonin gene (Amara et aL, 1982; Rosenfeld et al., 1983).
`discovered after its structure and existence were predicted from molecular cloning
`Calcitonin gene -related peptide (CORP) is a 37 -amino acid peptide which was
`
`1.1. Calcitonin gene -related peptide
`
`General introduction
`
`CHAPTER 1
`
`"
`
`" should read "A form of CGRP
`
`Correction (page 1, lires 24 -25): "A CGRP
`
`

`

`HaCGRP
`
`HßCGRP
`
`RaCGRP
`
`RßCGRP
`
`NH2
`
`NH2 -Ser
`
`NH2 -Ser
`
`Porcine CGRP NH2 -Ser
`
`Human amylin NI -I2 -Lys
`
`I
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`ID
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`NH2 -Ala Cys Asp Thr Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu Ser Arg Ser
`
`Asn
`
`Asn
`
`Asn
`
`Asn
`
`Asn
`
`Ala
`
`Gln
`
`Asn Phe
`
`Val His
`
`1
`
`2
`
`3
`
`.1
`
`6
`
`8
`
`9
`
`10
`
`Il
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`21
`
`22
`
`23
`
`25
`
`20
`
`24
`
`26
`
`27
`
`28
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`Gly Gly Val Val Lys Asn Asn Phe Val Pro Thr Asn Val Gly Ser Lys Ala Phe CONI-i2
`
`Met
`
`Ser
`
`Asp
`
`Asp
`
`Ser
`
`CONH2
`
`CONH2
`
`CONI -I2
`
`CONH2
`
`Glu
`
`Glu
`
`Asp
`
`HaCGRP
`
`HßCGRP
`
`RaCGRP
`
`RßCGRP
`
`Porcine CGRP
`
`Met
`
`Human amylin Ser Asn Asn Phe Gly Ala Ile Ile Ser Ser
`
`Asn Thr Tyr -CONH2
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`Figure 1.1: Structure of a and ß forms of CGRP from man, rat and pig. The structure of human amylin
`is shown for comparison. Broken horizontal lines indicate regions of sequence identity with HaCGRP.
`Cysteine residues at positions 2 and 7 (in bold) take part in a disulphide bond.
`
`CGRP and another 37 -amino acid peptide called amylin which is secreted from
`islets of Langerhans. Human amylin, also known as
`pancreatic
`islet amyloid
`polypeptide, was originally isolated from the amyloid deposits of an insulinoma
`(Westermark et al., 1986) and of Type II diabetic pancreases (Cooper et al., 1987). It
`has 43% and 46% sequence homology with HaCGRP and human ßCGRP ( HßCGRP)
`respectively. Indeed, amylin shares many of the biological properties of CGRP and
`cross -reacts with CGRP receptors (Section 1.1.9.1.).
`More recently, a novel 52 amino acid peptide showing slight sequence
`homology to CGRP has been isolated from human phaeochromocytoma (Kitamura et
`al., 1993). This peptide has been named adrenomedullin. Nine amino acid residues in
`the C- terminal (15 -52) end of adrenomedullin are also found in the a and ß forms of
`human CGRP.
`
`1.1.2. Distribution of CGRP
`
`CGRP is widely distributed in the central and peripheral nervous systems (Tschopp et
`al., 1984; Lee et al., 1985; Wimalawansa, et al., 1987). The presence of CGRP
`immunoreactivity has been detected primarily by
`immunocytochemistry and
`radioimmunoassay with anti -CGRP sera which do not distinguish between the two
`
`2
`
`

`

`known forms of CGRP. However, the expression of the a and ß forms of CGRP has
`been revealed by hybridization histochemistry using specific RNA probes (Mulderry
`et al., 1988; Noguchi et al., 1990; Sternini & Anderson, 1992). Both forms of CGRP
`are localized primarily in neural tissues.
`In the brain, CGRP is present in the nuclei of sensory and motor cranial nerves
`and in cell bodies in distinct regions including the hypothalamus, preoptic area,
`ventromedial thalamus, medial amygdala and hippocampus (Skofitsch & Jacobowitz,
`1985a; Yamamoto & Tohyama, 1989). In the spinal cord, CGRP -immunoreactive
`fibres are distributed primarily in the dorsal horn which receives sensory input. Dorsal
`rhizotomy induces a marked loss of CGRP -immunoreactive fibres from the dorsal
`spinal cord. Thus the CGRP -containing fibres are central projections of afferent
`neurons originating from the dorsal root ganglion (Gibson et al., 1984). The mRNAs
`of the a and ß forms of CGRP are co- expressed in dorsal root ganglion cells (Noguchi
`et al., 1990).
`CGRP immunoreactivity is found within cells and sensory nerve fibres in
`diverse peripheral organs including the heart, lung, urogenital tract, tongue, pancreas,
`skin and gastrointestinal tract (Gibbins et al., 1985; Wimalawansa et al., 1987;
`Mulderry et al., 1988). Throughout the body, it is localized in perivascular sensory
`nerve fibres (Rosenfeld et al., 1983; Mulderry et al.; 1985, Uddman et al., 1986). The
`density of fibres around arteries is generally higher than that around veins (Uddman et
`al., 1986). Combined retrograde tracing and immunocytochemical studies have
`demonstrated that the CGRP -immunoreactive nerves in the periphery originate from
`dorsal root ganglia (Alm & Lundberg, 1988; Louis et al., 1989; Sternini & Anderson,
`1992). However, the origin of most of the CGRP -immunoreactive cerebrovascular
`nerve fibres appears to be the trigeminal ganglion. CGRP -immunoreactive fibres are
`found in the adventitia and the adventitial- medial border of blood vessels (Gulbenkian
`et al., 1986; Edvinsson et al., 1987; Shoji et al., 1987).
`in all regions of the heart,
`CGRP -immunoreactive fibres are present
`particularly in association with the coronary arteries, within the papillary muscles,
`and within the sinoatrial and atrioventricular nodes (Mulderry et al., 1985). Tissue
`concentrations of immunoreactive CGRP are higher in the atria than the ventricles in
`rat and guinea pig hearts (Wharton et al., 1986; Wimalawansa & Maclntyre, 1988). In
`human cardiopulmonary tissue, the highest levels of CGRP immunoreactivity are
`found in the left anterior descending coronary artery, followed in declining order by
`the bronchus, right atrium, pulmonary artery, lung and left ventricle (Franco -
`Cereceda, 1991). CGRP -immunoreactive nerve fibres are very sparse in the proximal
`region of human epicardial arteries but increases in number distally (Gulbenkian et
`al., 1993).
`
`3
`
`

`

`CGRP immunoreactivity is also localized in non -nervous tissue. The peptide
`was originally reported to be absent in the rat thyroid gland (Rosenfeld et al., 1983).
`immunoreactivity has been subsequently co- localized with
`However, CGRP
`calcitonin in thyroid C -cells (Sabate et al., 1985; Lee et aL, 1985). Rat thyroid C -cells
`produce both calcitonin and CGRP mRNAs in a ratio of approximately 95:1. CGRP
`immunoreactivity is also localized in nerve fibres in the thyroid gland. In the lung,
`CGRP immunoreactivity is localized in capsaicin -sensitive nerve fibres and in
`endocrine cells (Cadieux et al., 1986; Shimosegawa & Said, 1991). CGRP
`immunoreactivity has been localized in subpopulations of endothelial cells of term
`human um