Design and Optimization of Recombinant Antibodies Directed Against Platelet Glycoprotein VI with ...

275 Pages·2011·25.24 MB·English

UNIVERSITÉ PARIS-SUD 11

ECOLE DOCTORALE :
INNOVATION THÉRAPEUTIQUE : DU FONDAMENTAL A L’APPLIQUÉ
PÔLE : PHYSIOPATHOLOGIE MOLECULAIRE ET CELLULAIRE

DISCIPLINE :
Physiopathologie cellulaire et moléculaire

ANNÉE 2011 - 2012 SÉRIE DOCTORAT N°

THÈSE DE DOCTORAT
soutenue le 24 novembre 2011

par
Muhammad ZAHID

Design and Optimization of Recombinant Antibodies Directed
Against Platelet Glycoprotein VI with Therapeutic and Diagnostic
Potentials

Directeur de thèse : Professeur Philippe BILLIALD, EA4530, Université Paris-Sud 11
Co-directeur de thèse : Docteur Martine JANDROT-PERRUS INSERM U698

Composition du jury :

Président du jury : Professeur Dominique PORQUET, Doyen de la Faculté de Pharmacie, Université Paris-Sud 11
Rapporteurs : Docteur Richard LE NAOUR, EA 4303, Université de Reims, Champagne-Ardenne
Professeur Philippe NGUYEN, EA 3801, Université de Reims, Champagne-Ardenne
Examinateurs : Professeur Philippe BILLIALD, Faculté de Pharmacie, Université Paris-Sud 11
Professeur Delphine BORGEL, EA4531, Faculté de Pharmacie, Université Paris-Sud 11
Docteur Martine JANDROT-PERRUS, Inserm U698, Hôpital Bichat, Paris

ACKNOWLEDGEMENT

I would like to begin by thanking Professor Philippe BILLIALD for his super-human
patience and valuable advice. Thank you for standing by me at all tough times and for being
part of the great moments of my PhD. Thank you, for your vision and for teaching me the
intricacies and the beauty of the recombinant antibody technology. Once again a very warm
‘thank you’ to gave a naive pakistani boy a chance to begin his journey through molecular
biology, for introducing me to the world of science and for instilling in me the spirit of the
free scientist.
I have no appropriate words to manifest my feeling of respect, gratefulness and
obligations for Dr. Martine JANDROT-PERRUS for providing constant guidance, useful and
timely suggestions and encouragement throughout my research work. She really took great
interest in my whole research work. Thanks, for your advice on a million things, for teaching
me how to be pragmatic in research, for innumerable interesting discussions… I could go on.
I am still waiting for my quiche!
I thank Professor Dominique PORQUET and Professor Delphine BORGEL for
accepting to be part of my jury. I would like to express my utmost gratitude to Professor
Philippe NGUYEN and Dr. Richard LE NAOUR for their valuable comments and discussions
on my thesis.
I am thankful to Professor Jean-Baptiste MICHEL, Professor Cécile BERNARD and
Professor Christian POÜS who opened their labs to me and who taught me that hard work
does pay off!
I am also greatly thankful to Professor Max GOYFFON for his valuable and thought
provoking advices.
Thank you Professor Isabelle DIMIER-POISSON for providing all the possible
facilities in your lab during my master and Dr. Nicolas AUBREY for your help throughout
my research work for especially recombinant antibody fragments purification.
A successful thesis in biology is never accomplished alone. I have been extremely
lucky to be surrounded by many bright and caring people. Stéphane LOYAU, you are one in
a million. Thank you for holding my hand during my work in the lab, for teaching me the
basics of laboratory life and for listening to my cribbing!
Maxime BOUABDELLI, we started this long journey together, and now we are each
heading in separate directions. Thanks for always staying beside me, through the good times
and the worst! Thanks for helping me out in many experiments.
Thanks Rana IFTIKHAR for many great moments, for being thoughtful and thanks for
listening to my million cribs and for being an excellent roommate! Good luck for a great PhD.
Thank you Marie Christine BOUTON, Véronique AROCAS, Véronique OLLIVER
Laurence VENISSE, Claude YEPREMIAN and Magali NOIRAY for your advice on
everything.
Finally, I thank my loving family without whom I would not be here today. Lastly I
would like to mention the name of my son Khizar ZAHID, whose sweet memories always
remain in my heart.

I thank everyone involved in the work presented in this thesis.
Table of Contents

PART A: STATE OF THE ART

1 Historical Development of Therapeutic Antibodies...........................................1
1.1 Antibodies Engineering...............................................................................6
1.1.1 Structure of Ig Immunoglobulin..........................................................6
1.1.2 Antibodies in Bodies............................................................................7
1.1.3 Murine Monoclonal Antibodies Production........................................8
1.1.4 Chimerization.....................................................................................10
!"!"# Humanization.....................................................................................12
1.1.6 Alternatives........................................................................................16
1.2 Fully Human Antibodies...........................................................................17
1.2.1 Human Monoclonal Antibodies.........................................................17
1.2.2 Different Approaches of Human Monoclonal Antibodies Prod........18
1.2.3 Human B Cell Hybridoma.................................................................18
1.3 Antibodies Selecting Methods..................................................................20
1.3.1 Exposure on the Surface of Phage (Phage Display)..........................21
1.3.2 Exposure on the Surface of a Ribosome (Ribosome Display)...........23
1.3.3 Exposure on the Yeast Surface (Yeast Display)................................24
1.4 Design of Novel Antibody Formats with Antigen-binding Activity.........24
1.4.1 Monovalent Antibody Formats..........................................................24
1.4.2 Polyvalent Antibody Fragments........................................................34
1.4.3 Armed Antibodies (Cancer/Immunoconjugates)...............................38
1.4.4 Antibody Fragment Expression System.............................................39
2 Platelets, Thrombosis and Anti-Thrombotics...................................................49
2.1 Hemostasis and Thrombosis......................................................................49
2.1.1 Major Types of Thrombosis...............................................................51
2.1.2 Different Phases of Thrombus Formation..........................................52
2.1.3 Stabilization Phase or Perpetuation...................................................53
2.1.4 Actors Involved in Thrombus Formation..........................................53
2.2 Platelets.....................................................................................................58
$"$"! Platelet Structure................................................................................59
I
2.2.2 Platelet Plug Formation......................................................................65
2.2.3 Other Functions of Platelets besides Hemostasis...............................70
2.3 Platelet Receptors: Structure and Function...............................................71
2.3.1 The GPIb/V/IX Complex and vWF...................................................71
2.3.2 Integrins.............................................................................................74
2.3.3 Collagen Receptors............................................................................76
2.3.4 G-Protein-Coupled Receptors (GPCRs)............................................78
2.3.5 Recently Identified Receptors Stabilizing Thrombosis.....................82
2.4 Antithrombotic Drugs...............................................................................84
2.4.1 Anticoagulants...................................................................................84
2.4.2 Antiplatelet Agents............................................................................86
2.4.3 New Class of Antiplatelet Agents in Clinical Trials..........................97
2.5 Adhesion : Target of the Future ?..............................................................99
2.5.1 Inhibiting Platelet Adhesion..............................................................99
2.5.2 GPIb-VWF Axis..............................................................................100
2.5.3 Integrin !2"1 and GPVI...................................................................105
2.6 GPVI, a Major Receptor of Collagen......................................................106
2.6.1 Nucleotide Sequence, Genomic Structure of GPVI and
Polymorphisms..................................................................................................107
2.6.2 GPVI Ligands..................................................................................109
2.6.3 Structure of GPVI............................................................................111
2.6.4 GPVI Coupled Signaling Pathway...................................................116
2.6.5 GPVI Down-regulation....................................................................118
2.6.6 GPVI Deficiencies...........................................................................120
2.6.7 GPVI in Haemostasis and Thrombosis............................................123
2.6.8 GPVI and Inflammation...................................................................125
2.6.9 Strategies for Therapeutic Targeting of GPVI.................................125

PART B: EXPERIMENTAL APPROACHES

3 Aim of the study.............................................................................................131
3.1 Design of Anti-GPVI scFv 9O12 with Diagnostic Potential..................133
3.2 Design of Anti-GPVI Recombinant Antibody Fragments with Therapeutic
Potential .................................................................................................................135
II
3.2.1 Redesigning of a humanized Single-chain Fv Directed Against
Human Platelets GPVI.......................................................................................137
3.2.2 Recombinant Antibody Fab Fragments...........................................138

PART C: GENERAL DISCUSSION AND PERSPECTIVES

4 General Discussion and Perspectives.............................................................143

REFERENCES 146

ANNEXES 179

Annexe 1:
Scorpion antivenoms: Progresses and challenges.
M. ZAHID, S. ADI-BESSALEM, J. MUZARD, M. JUSTE, M.F. MARTIN-EAUCLAIRE, N.
AUBREY, F. LARABA-DJEBARI, P. BILLIALD.
In J. Barbier, E. Benoit, P. Marchot, C. Mattei, D. Servent (Eds), E-Book Rencontres en
Toxinologie 18 « Advances and new technologies in Toxinology ». Société française pour l'Etude des
Toxines. 2010, pp 243-252.

Annexe 2:
Design and humanization of a murine scFv that blocks human platelt
glycoprotein VI in vitro.
J. MUZARD, M. BOUABDELI, M. ZAHID, V. OLLIVIER, J.J. LACAPERE, M. JANDROT-
PERRUS, P. BILLIALD.
FEBS Journal. 2009, 276, 4207-4222.
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1 Historical Development of Therapeutic Antibodies

Among the marvels of scientific research which have distinguished our century, no
achievements are more remarkable, nor of greater moment to the welfare of mankind,
than those pertaining to the field of biology, pathology and therapeutic investigation.
A glance at the history of therapeutic procedure in the prophylactic treatment of
infectious diseases shows that the general principle underlying all later discoveries
had been, though crudely, divined at a much earlier period than we are wont to
suppose. It is known that the ancient Hindus and Persians, as well as the nomad tribes
and caravans of farther Asia, practised inoculation of equine virus or horse-pox - the
mammary pustule developed during early lactation in the horse, camel, cow and even
in woman. The inoculation of human virus is of immemorial origin, probably coeval
with the importation of variola from Asia into Africa by the Saracens. It is certain that
as early as the tenth century the Arabs and Chinese adopted the custom of
variolization (inoculation of small-pox).
It was Jenner (1776), who started the systematic and exhaustive study of the subject
destined to prove inestimably beneficial to mankind and then Pasteur who, in 1880,
announced to the world the issue of his labors, touching the protective inoculation of
animals and thus was the broken thread of pathogenic research taken up a new and the
task of solving its mysteries resumed - be it said with more profound acumen and far
more complete appliances than ever before.
The French savant demonstrated that cultures of the bacilli of chicken-cholera,
when thoroughly dried and long exposed to the air, lost their virulence and that fowls
inoculated with the attenuated virus were rendered insensible to the attacks of more
energetic micro-organisms. It was, mutatis mutandis, a modification or development
of the Jennerian principle: "The history of vaccination constitutes the first step in a
long series of labours inspired by the admirable discoveries originating in the genius
of Pasteur. The principle is always the same - to diminish the strength of the virus and
inject it into the animal which we wish to render immune "(Bernheim). Salmon and
Smith, in 1886-87, showed conclusively that animals may be rendered immune
against certain infectious diseases by inoculating them with filtered cultures
containing the toxic products of pathogenic micro-organisms entirely free from the
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living bacteria to which they owe their origin. By this process immunity against the
bacillus of hog-cholera was attained in pigeons.
Roux (1888), employing similar sterilized cultures, succeeded in protecting
susceptible animals against the anthrax bacillus (Roux and Yersin, 1888); and then
Behring and Kitasato (1890) have proved that immunity against the action of the
tetanus bacillus may be conferred by the use of toxic products in solution free from
the presence of active germs ( von Behring and Kitasato, 1890, von Behring and
Kitasato 1991b). The significance of this discovery could hardly be over-estimated.
By it the entire theory of causal phenomena- the protective force in which the
immunizing property was supposed to reside - became modified. If not a living
organism, but a chemical substance, proved to be the immunizing agent, then
resistance to toxic influences must proceed from some source other than bacterial
metabolism - some organic force inherent in the inoculated system. To ascertain the
nature and operation of this bactericidal power and determine the rationale of
acquired immunity now engaged the earnest attention of savants throughout the
world.
Despite use of the antitoxin, death rates from diphtheria were still high in the early
1900s and the need for a vaccine was clear. In 1913, von Behring had produced long
lasting immunity in guinea pigs, monkeys and asses using a carefully balanced
mixture of toxin and antitoxin ( Von Behring and Kitasato, 1913). This was used in
the first vaccination studies on humans. A widespread immunization program
followed the development of formalin-inactivated toxin by A. T. Glenny and Barbara
Hopkins (Glenny and Hopkins, 1923) and Gaston Ramon in the early 1920s. The
antitoxin was first used to treat a seriously ill girl in 1891, who subsequently
recovered.
Production of the antitoxin on a large scale was achieved in horses, with both the
diphtheria serum and the antiserum being standardised using guinea pigs. Widespread
use of the antitoxin followed and studies in rabbits showed that it had to be
administered soon after infection to be effective (Zinsser, 1931).
Anti-infectious serotherapy was used for the first time in humans in 1891, followed
by the development of antivenom by Phisalix and Calmette (1894). The term antibody
(antikörfs) was coined by Pfeiffer in 1898 later ‘magic bullet’ by Paul Ehrlich (1908).
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