Detection and Prediction of Factor VIII Antibody Formation in Congenital and Acquired ...

258 Pages·2017·3.13 MB·English

Detection and Prediction of Factor VIII Antibody
Formation in Congenital and Acquired
Haemophilia A

Paul Andrew Batty
BSc(Hons) MB BS MRCP

Submitted in partial fulfilment of the requirements of the
Degree of Doctor of Philosophy

Centre for Immunobiology
Institute of Cell and Molecular Science
Barts and the London School of Medicine and Dentistry
Queen Mary University of London
London, E1 2AT

January 2016

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Statement of Originality

I, Paul Andrew Batty, confirm that the research included within this thesis is my own work or
that where it has been carried out in collaboration with, or supported by others, that this is
duly acknowledged below and my contribution indicated. Previously published material is
also acknowledged below.
I attest that I have exercised reasonable care to ensure that the work is original, and does
not to the best of my knowledge break any UK law, infringe any third party’s copyright or
other Intellectual Property Right, or contain any confidential material.
I accept that the College has the right to use plagiarism detection software to check the
electronic version of the thesis.
I confirm that this thesis has not been previously submitted for the award of a degree by this
or any other university.
The copyright of this thesis rests with the author and no quotation from it or information
derived from it may be published without the prior written consent of the author.

Signature:

Date: 24th January 2016

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Details of Collaborations

Testing of clinical samples (Nijmegen-Bethesda assay and Factor VIII ELISA) as part of
Chapters 6 and 7 were performed as part of routine care within specialist haemostasis
laboratories at The Royal London Hospital and St Thomas’ Hospital. I designed and performed
all analyses within these studies. I performed all subsequent experimental work optimising
pre-analytical heat treatment incubation conditions.
The epitope mapping work described in Chapter 8, involved a collaboration with the
Department of Biological Sciences and Institute of Molecular and Structural Biology,
Birkbeck, University of London (Dr Adrian Shepherd, Reader in Computational Biology and
Stuart Skelton, PhD Candidate) and the Angelo Bianchi Bonomi Hemophilia and Thrombosis
Centre, Milan, Italy (Prof Flora Peyvandi, Dr Elisa Mancuso and Dr Elena Santagostino). Local
approvals were obtained and clinical samples collected by Dr Elisa Mancuso and her team.
Samples were robotically loaded onto the Pepscan array by staff within Pepscan, Lelystad,
Netherlands. Normalisation of solvent accessibility, distances between the alpha carbons and
threading within pGENTHREADER were performed by Stuart Skelton. I designed, performed
all analyses and interpreted all structural data from these experiments.
RNA extractions within Chapter 9, were performed within the Genome Centre Core Facility
Charterhouse Square, London. I designed this study, collected all samples and performed all
data analyses.
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Details of Publications

The following publications have arisen from this thesis:
1) Batty P, Platton S, Bowles L, Pasi KJ, Hart DP. Pre-analytical heat treatment and a
FVIII ELISA improve Factor VIII antibody detection in acquired haemophilia A. Br J
Haematol 2014; 166(6):953-956.
2) Batty P, Palmer B, Chalmers E, Hay CR, Liesner R, Rangarajan S et al. A national
survey of immunosuppression strategies for acquired haemophilia A. Haemophilia
2015; 21(1):e73-e76.
3) Batty P, Moore GW, Platton S, Maloney JC, Palmer B, Bowles L et al. Diagnostic
accuracy study of a factor VIII ELISA for detection of factor VIII antibodies in
congenital and acquired haemophilia A. Thromb Haemost 2015; 114(4):804-811.

The following abstracts (poster, P and oral communications, OC) have arisen from this thesis:
1) Batty P, Skelton S, Shepherd AJ, Hart DP. Amino acid sequence epitope mapping of
four factor VIII monoclonal antibodies. J Thromb Haemost 2013; 11(Supplement
S2):579. ISTH, Amsterdam, 2013 (P)
2) Batty P, Moore GW, Platton S, Maloney JC, Bowles L, Pasi J et al. Immunological
detection of factor VIII antibodies in congenital and acquired haemophilia A. J
Thromb Haemost 2013; 11(Supplement S2):930. ISTH, Amsterdam, 2013 (P)
3) Batty P, Platton S, Hart DP. The Bethesda assay in isolation significantly
underestimates the presence of factor VIII antibodies in acquired haemophilia A. Br
J Haematol 2013; 161(Supplement S1):13. BSH, Liverpool, 2013 (OC)
4) Batty P, Rangarajan S, Austin S, Khair K, Millar C, Stümpel J et al. Uptake and timing
of inhibitor screening in non-severe hemophilia A: results of a pan-London
evaluation. Haemophilia 2014; 20(Supplement S3):48. WFH, Melbourne, 2014 (P)
5) Batty P, Collins PW, Chalmers E, Hay CR, Liesner R, Palmer B et al. A UKHCDO National
Survey of the management of acquired haemophilia A. Haemophilia 2014;
20(Supplement S2):31. EAHAD, Brussels, 2014 (P)
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6) Batty P, Skelton S, Shepherd AJ, Hart DP. Amino acid sequence epitope mapping of
anti-factor VIII monoclonal antibodies. Br J Haematol 2014; 165(Supplement S1):8.
BSH, Birmingham, 2014 (OC)
7) Batty P, Skelton S, Shepherd AJ, Mancuso ME, Santagostino E, Peyvandi F et al. High
throughput amino acid sequence epitope mapping of inhibitory antibodies in severe
hemophilia A. J Thromb Haemost 2015; 13(Supplement S2):135. ISTH, Toronto, 2015
(OC)
8) Batty P, Mein C, Wozniak E, Hart DP. Reliable RNA retrieval from low-volume paxgene
tubes in simulated "real world" next generation sequencing (RNA-SEQ): Gena-05
substudy pilot. J Thromb Haemost 2016; 13(Supplement S2):365. ISTH, Toronto, 2015
(P)
9) Batty P, Platton S, Hart DP. Optimisation of Pre-Analytical Heat Treatment for Factor
VIII Antibody Detection. Haemophilia 2016; 22(Supplement S2):88. EAHAD, Malmö,
2016 (P)
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Abstract
Factor VIII (FVIII) is a co-factor in the haemostatic system required for fibrin-rich clot
formation. Inherited F8 gene defects result in haemophilia A (HA), one of the commonest
inherited bleeding disorders. Acquired FVIII defects (acquired haemophilia A, AHA) occur
through auto-antibody formation. FVIII antibodies (allo and auto-antibodies) are the greatest
challenges facing the haemophilia treating physician. Prediction of risk of antibody formation
is based on genetic and environmental factors. There is incomplete understanding of the
total immune response to FVIII due to limitations in current laboratory methodology used for
FVIII antibody testing.
The aim of this this work was to assess clinical practices, laboratory methodology and high-
throughput approaches to further characterise the immune response to FVIII. The key
findings are as follows:
1) Sub-optimal compliance with targeted inhibitor screening following FVIII treatment
was seen in non-severe HA in London haemophilia centres.
2) A national survey of AHA demonstrated heterogeneity in the management of
immunosuppression and testing.
3) A FVIII ELISA was specific with a high negative predictive values for FVIII antibody
detection in routine practice.
4) Pre-analytical heat treatment prior to antibody testing, improved sensitivity for auto-
antibody detection and a systematic evaluation optimised incubation conditions for
this modification.
5) A novel re-usable high-throughput peptide microarray, characterised B-cell epitopes
of monoclonal and polyclonal FVIII antibodies, demonstrating immunodominant
epitopes in regions of functional or structural importance.
6) A modified low volume RNA sample tube demonstrated feasibility for transcriptome
analysis in patients with severe haemophilia A, providing a repository of
transcriptome data for developing understanding of the allo-immune response to
FVIII.
Heterogeneity in clinical and laboratory practices limits interpretation of data from
observational studies of FVIII antibodies. Improvements in detection and characterisation of
FVIII antibodies, may further advance understanding of the total immune response to FVIII
identifying biomarkers for risk stratification.
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Dedication

This thesis is dedicated to Sofia, Alex, Cristina, Angela and Philip
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Acknowledgements
I am grateful to my supervisors Dan Hart and Adrian Newland for the opportunity to pursue
my research interests in haemophilia. Dan’s support and tireless energy has been truly
inspirational. The support and tutorage of Sean Platton has been invaluable guiding me from
having little laboratory experience, allowing me to develop both technical skills and a
scientific approach. Sean and his teams’ support during the months of optimising the pre-
analytical heat treatment conditions, made long hours sitting in front of a water-bath fly by.
I am grateful to Prof John Pasi for his encouragement which has nurtured both my academic
and writing interests. Working as part of the team at The Royal London Hospital haemophilia
centre has been invaluable and I will miss each member as I move back into clinical work.
I am hugely fortunate to have had the opportunity to collaborate with a number of excellent
academic units both nationally and internationally. The Computational Biology team at
Birkbeck University, in particular Adrian Shepherd and Stuart Skelton have provided endless
encouragement for the epitope mapping work. Stuart has taught me how to use many in-
silico methodologies and always found time to explain these simply. The UKHCDO Inhibitor
Working Party, in particular Prof Peter Collins, has been instrumental in the development of
the national survey presented in this work and also the development of ideas surrounding
inhibitor testing. Collaboration with the team working at the Milan haemophilia centre, in
particular Flora Peyvandi and Marcin Gorski, on the B-cell epitope mapping work has been
an amazing experience. I am also grateful for the teams at all of the London haemophilia
centres for their help with data collection within the Pan-London audit. The support and
statistical guidance offered by Ben Palmer at the National Haemophilia Database has been
invaluable and I am grateful for the time he has taken to guide me over the last few years. I
am grateful for the support of Chaz Mein and his team at the Genome Centre who have
guided me through the RNA-Seq work. I am also grateful to all of the volunteers and patients
who provided samples, with whom none of this work would have been possible.
I am grateful for the support offered by Octapharma, both in the funding of this fellowship
and for having the forward vision for inclusion of the RNA-Seq work within the GENA-05
study.
Finally, this work would not have been possible without the support, love and patience
provided by my family from Sofia, Alex, Cristina and my parents who have allowed me to
pursue these dreams.
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List of Abbreviations
aa Amino acids
ABR Annualised bleed rate
AHA Acquired haemophilia A
ANOVA Analysis of variance
APC Activated Protein C
aPTT Activated partial thromboplastin time
ASA Accessible surface area
ASGRP Asialoglycoprotein receptor
BCR B-cell receptor
BDD B domain deleted
bFVIII:C Baseline FVIII:C
BHK Baby hamster kidney
bp Base pairs
BSA Bovine serum albumin
CANAL Concerted action on neutralizing antibodies in severe haemophilia
CBA Classical Bethesda assay
CCC Comprehensive care centre
CCL3L1 Chemokine (C-C motif) ligand 3-like 1
CCL3L3 Chemokine (C-C motif) ligand 3-like 3
CD Cluster of differentiation
ChBA Chromogenic Bethesda assay
CHO Chinese hamster ovary
CI Confidence interval
CLEC4M C-type lectin domain family 4 member M
CLIPS Chemical linkage of peptides onto scaffolds
CPA Clinical pathological accreditation
CR Complete response
CTLA4 Cytotoxic T-lymphocyte-associated protein 4
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CXCL7 Chemokine (C-X-C motif) ligand 7
CXCL9 Chemokine (C-X-C motif) ligand 9
CXCL11 Chemokine (C-X-C motif) ligand 11
DC Dendritic cell
DDAVP 1-deamino-8-d-arginine vasopressin
Del Deletion
DNA Deoxyribonucleic acid
DOR Diagnostic odds ratio
DSSP Dictionary of protein secondary structure
EACH2 European acquired haemophilia registry
ECAT European concerted action on thrombosis foundation
ED Exposure days
ELISA Enzyme linked immunosorbent assay
EQA External quality assurance
EUHASS European haemophilia safety surveillance
Ex Exon
F8DP Factor VIII deficient plasma
Fab Fragment antigen binding
Fc Fragment crystallisable region
FEIBA Factor VIII inhibitor bypassing activity
FLI Fluorescence-based immunoassays
FMOC Fluorenylmethyloxycarbonyl
FVIII Factor VIII
FVIII(a) Factor VIII (activated)
FVIII:C Factor VIII coagulant activity
FIX(a) Factor IX (activated)
FX(a) Factor X (activated)
GEE Generalised estimating equations
GTH Gesellschaft für thrombose-und hämostaseforschung
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