Sequence and structural analysis of antibodies

250 Pages·2009·2.71 MB·English

UNIVERSITY COLLEGE LONDON
UCL Research Department of Structural and Molecular Biology
Sequence and structural analysis of
antibodies
Abhinandan K. Raghavan
A dissertation submitted to University College London for the
degree of Doctor of Philosophy
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Declaration
I, Abhinandan K. Raghavan, con(cid:12)rm that the work presented in this thesis is my
own. Where information has been derived from other sources, I con(cid:12)rm that this
has been indicated in the thesis.
Abhinandan K. Raghavan
April 5, 2009
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Abstract
The work presented in this thesis focusses on the sequence and structural analysis
of antibodies and has fallen into three main areas.
First I developed a method to assess how typical an antibody sequence is of the
expressed human antibody repertoire. My hypothesis was that the more \human-
like" an antibody sequence is (in other words how typical it is of the expressed
human repertoire), the less likely it is to elicit an immune response when used
in vivo in humans. In practice, I found that, while the most and least-human
sequences generated the lowest and highest anti-antibody reponses in the small
available dataset, there was little correlation in between these extremes.
Second, I examined the distribution of the packing angles between V and V
H L
domains of antibodies and whether residues in the interface in(cid:13)uence the packing
angle angle. This is an important factor which has essentially been ignored in
modelling antibody structures since the packing angle can have a signi(cid:12)cant e(cid:11)ect
on the topography of the combining site. Finding out which interface residues
have the greatest in(cid:13)uence is also important in protocols for ‘humanizing’ mouse
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antibodies to make them more suitable for use in therapy in humans.
Third, I developed a method to apply standard Kabat or Chothia numbering
schemes to an antibody sequence automatically. In brief, the method uses pro(cid:12)les
to identify the ends of the framework regions and then (cid:12)lls in the numbers for each
section. Benchmarking the performance of this algorithm against annotations in
the Kabat database highlighted several errors in the manual annotations in the
Kabat database. Based on structural analysis of insertions and deletions in the
framework regions of antibodies, I have extended the Chothia numbering scheme
to identify the structurally correct positions of insertions and deletions in the
framework regions.
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Abbreviations
The following abbreviations have been used in this thesis:
Amino acids
One-letter Three-letter Amino-acid
code code
A Ala Alanine
C Cys Cysteine
D Asp Aspartic acid
E Glu Glutamic acid
F Phe Phenylalanine
G Gly Glycine
H His Histidine
I Ile Isoleucine
K Lys Lysine
L Leu Leucine
M Met Methionine
N Asn Asparagine
P Pro Proline
Q Gln Glutamine
R Arg Arginine
S Ser Serine
T Thr Threonine
V Val Valine
W Trp Tryptophan
Y Tyr Tyrosine
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Miscellaneous
ANN Arti(cid:12)cial Neural Network
BLAST Basic Local Alignment and Search Tool
GA Genetic Algorithm
NH Number of hidden nodes in neural network
NN Neural Network
PDB Protein Data Bank
Rprop Resilience propogation
SNNS Stuggart Neural Network Simulator
SRS Sequence Retrieval Service
SSE Sum of Square Error
SSEARCH Sequence Search
SVM Support Vector Machine
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Acknowledgements
At the outset, I would like thank my supervisor, Dr. Andrew Martin, for the
support and patience through what has been a very exciting PhD. I couldn’t
thank him enough for all the help that he has extendend to me.
I’d like to thank members of the group, Anja, Craig, Jacob, Lisa, and Sri for being
so nice and helpful throughout and for making the lab such a wonderful place to
work in. I would also like to thank my mentor, Prof. Christine Orengo, for her
encouragement and help, and the BBSRC and GlaxoSmithKline for funding my
PhD. I am very grateful to Tom, Jesse, and Jahid for excellent maintenance of the
computers and clusters in the department. I have used the research computing
resources at UCL extensively over the last couple of years and the help of all sta(cid:11)
who maintain these resources is gratefully acknowledged.
This PhD would never have been possible without the support of my family,
particularly my wife Kalyani. I’d like to thank her for being a wonderful partner,
teacher, andapillarofsupportduringexactingphases ofmyPhD.Iwouldalsolike
to thank my parents, sister, and my wife’s family for being extremely supportive
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during the last 4 years.
I’ve made innumerable friends in UCL and in London, each of whom has been
very special. Making it this far is unimaginable without them. I’d like to thank
them all - Lisa, Andrew, Jacob, Anja, Sri, Craig, Sunita, Roger, Juan, Ali, Sarah,
Tom, Jesse, Duncan, Jahid, Kanchan, Ranga, Padu, Natesh, Mark, Sanjay, An-
war, Gillian, Tjelvar, Michael, Antonio, Stefano, Tom, Paul, Dhami, Venu, Pape,
Harkamal, Meena, and the CATH team. In particular, I’d like to thank Sunita
and Roger for their technical inputs while I was writing grant proposals, Lisa for
her limitless patience in answering all my queries and her help, Michael Wright
for help with all the paperwork during my trips outside of the UK, Padu for being
a wonderful host when I needed a change from my cooking, and my cousin Ranga
and his wife Jyothi who made all holidays delightful. I’d also like to thank sta(cid:11)
at the numerous restaurants and eateries in London that made my life as a PhD
student a lot easier than I could imagine.
Dedication
This work is dedicated to my family, my home India, a country of extraordinary
uniqueness, and to London, my second home.
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Contents
Declaration 2
Abstract 3
Abbreviations 5
Acknowledgements 7
1 Introduction to immunology 26
1.1 Innate immune system . . . . . . . . . . . . . . . . . . . . . . . . . 27
1.2 The Adaptive Immune system . . . . . . . . . . . . . . . . . . . . . 29
1.2.1 B-Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . 30
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1.2.2 T-Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . 31
1.2.3 MHC molecules . . . . . . . . . . . . . . . . . . . . . . . . . 31
1.3 Activation of the adaptive immune system . . . . . . . . . . . . . . 32
1.3.1 Structure of an antibody . . . . . . . . . . . . . . . . . . . . 34
1.3.2 Generation of antibody diversity . . . . . . . . . . . . . . . . 35
1.3.3 VDJ Recombination . . . . . . . . . . . . . . . . . . . . . . 37
1.3.4 B-cell maturation, activation and proliferation . . . . . . . . 40
1.3.5 B-cell activation . . . . . . . . . . . . . . . . . . . . . . . . . 45
1.3.6 B-cell e(cid:11)ector-response . . . . . . . . . . . . . . . . . . . . . 48
1.4 T-cell responses and cell-mediated immune system . . . . . . . . . . 49
1.4.1 T-cell receptor . . . . . . . . . . . . . . . . . . . . . . . . . . 49
1.4.2 T-cell maturation . . . . . . . . . . . . . . . . . . . . . . . . 49
1.4.3 T-cell activation . . . . . . . . . . . . . . . . . . . . . . . . . 51
1.4.4 T-cell di(cid:11)erentiation . . . . . . . . . . . . . . . . . . . . . . 52
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