Immune responses in patients with Lysosomal Storage ...

Immune responses in patients with

Lysosomal Storage Disorders treated with

Enzyme Replacement Therapy and

Haemopoietic Stem Cell Transplantation

A thesis submitted to the University of Manchester for the degree of

Doctor of Medicine in the Faculty of Medical and Human Sciences

2012

Muhammad A Saif

MBBS, MRCP, FRCPath

School of Medicine

2

CONTENTS

1. Introduction ........................................................................................................................ 16

1.1. Lysosomal storage disorders ................................................................................... 17

1.2. Pathophysiology of LSD ............................................................................................ 18

1.2.1. Lysosomes and lysosomal enzymes ............................................................... 18

1.2.2. Cofactors for lysosomal enzymes .................................................................... 20

1.2.3. Effect of lysosomal enzyme deficiency ........................................................... 21

1.3. Clinical manifestations .............................................................................................. 24

1.4. Treatment strategies .................................................................................................. 27

1.4.1. Haemopoietic stem cell transplant (HSCT) ..................................................... 27

1.4.2. Enzyme replacement therapy (ERT) ................................................................. 37

1.4.3. Substrate reduction therapy (SRT) and chaperone mediated therapy (CMT)

42

1.4.4. Gene therapy ...................................................................................................... 44

1.5. Limitations of treatments .......................................................................................... 45

1.5.1. Limitations of HSCT ........................................................................................... 45

1.5.2. Limitations of ERT.............................................................................................. 47

1.5.3. Human immune responses to recombinant proteins ..................................... 48

1.5.4. Immune response to ERT .................................................................................. 50

1.5.5. Functional and clinical impact of immune response ..................................... 52

1.5.6. Effect of severity of immune response ............................................................ 53

1.5.7. Assays for detection and quantification of immune response ..................... 55

1.5.8. Limitations of existing assays .......................................................................... 57

1.6. Diagnosis, monitoring and disease biomarkers ..................................................... 57

1.7. Immune tolerance induction ..................................................................................... 58

1.8. Project aims ................................................................................................................ 63

1.8.1. Development of quantitative and functional immune assays for LSDs ....... 63

3

1.8.2. To study the incidence and impact of immune response in MPSIH patients

treated with ERT and HSCT ............................................................................................... 64

1.8.3. Evaluate the nature and incidence of immune response in other LSDs ...... 64

2. Materials and methods ...................................................................................................... 65

2.1. MPSI ............................................................................................................................. 71

2.1.1. Patients and sample preparation...................................................................... 66

2.1.2. IgG ELISA ............................................................................................................ 71

2.1.3. Enzyme activity assay ....................................................................................... 72

2.1.4. Mixing assay (quantification of catalytic inhibition) ....................................... 73

2.1.5. Cellular uptake inhibition .................................................................................. 75

2.2. Pompe diseas ............................................................................................................. 76

2.2.1. ELISA ................................................................................................................... 76

2.2.2. Enzyme assay in 96 well plate .......................................................................... 76

2.2.3. Catalytic inhibition assay .................................................................................. 77


2.3. MPS VI ......................................................................................................................... 79

2.3.1. ELISA ................................................................................................................... 79

2.3.2. Catalytic inhibition assay .................................................................................. 81


2.4. MPS II ........................................................................................................................... 84

2.4.1. ELISA ................................................................................................................... 84

2.4.2. Enzyme assay in 96 well plate .......................................................................... 84

2.4.3. 2.4.3 Catalytic inhibition assay ......................................................................... 86

2.4.4. .................................................................................................................................... 87

2.5. Western blot: .............................................................................................................. 87

2.6. Biomarkers and chimerism analysis ........................................................................ 88

2.7. Statistics ..................................................................................................................... 89

3. Development of quantitative and functional immune assays for Lysosomal Storage

Disorders..................................................................................................................................... 90

4

3.1. Introduction ................................................................................................................ 91

3.2. MPSI ............................................................................................................................. 92

3.2.1. ELISA ................................................................................................................... 92

3.2.2. Catalytic enzyme assay ..................................................................................... 95

3.2.3. Cellular uptake inhibition ................................................................................ 105

........................................................................................................................................... 107

3.3. Pompe disease ......................................................................................................... 108

3.3.1. ELISA ................................................................................................................. 108

3.3.2. Catalytic inhibition ........................................................................................... 111


3.4. MPS VI ....................................................................................................................... 120

3.4.1. ELISA ................................................................................................................. 120



3.5. MPSII .......................................................................................................................... 129

3.5.1. ELISA ................................................................................................................. 129


3.6. Discussion ................................................................................................................ 133

4. Haemopoietic stem cell transplantation ameliorates the high incidence of

neutralizing allo-antibodies observed in MPSI-Hurler after pharmacological enzyme

replacement therapy. ............................................................................................................... 137

4.1. Introduction .............................................................................................................. 138

4.2. Patient demographics .............................................................................................. 139

4.3. Pattern of immune response................................................................................... 142

4.3.1. ELISA ................................................................................................................. 142

4.4. Enzyme neutralization by antibodies ..................................................................... 146

4.4.1. Inhibition of cellular uptake of catalytically active enzyme ......................... 149

4.5. Immune response and disease biomarkers .......................................................... 151

4.6. Discussion ................................................................................................................ 154

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5. Incidence and functional nature of allo-antibodies in MPSI patients on long term ERT

158

5.1. Introduction .............................................................................................................. 159

5.2. Patient demographics .............................................................................................. 160

5.3. Allo-antibodies persist in some patients on long term ERT ................................ 161

5.4. Catalytic inhibition ................................................................................................... 164

5.5. Cellular uptake inhibition ........................................................................................ 166

5.6. Discussion ................................................................................................................ 169

6. Allo immune response in other Lysosomal Storage Disrders .................................... 172

6.1. Introduction .............................................................................................................. 173

6.2. Pompe diseas ........................................................................................................... 174

6.2.1. ELISA ................................................................................................................. 174

6.2.2. Catalytic inhibition assay ................................................................................ 176

6.2.3. Cellular uptake inhibition assay ..................................................................... 178

6.3. MPS VI ....................................................................................................................... 179

6.3.1. ELISA ................................................................................................................. 179

6.3.2. Catalytic inhibition assay ................................................................................ 181

6.3.3. Cellular uptake inhibition assay ..................................................................... 182

6.3.4. Correlation between antibody titre and biomarker ....................................... 183

6.4. Discussion ................................................................................................................ 184

7. Conclusions and Future direction .................................................................................. 187

7.1. Aim1: Development of quantitative and functional immune assays for LSDs .. 188

7.2. Aim 2: To study the incidence and impact of immune response in MPSI patients

treated with ERT and HSCT................................................................................................. 190

7.3. Aim 3: Evaluate the nature and incidence of immune response in other LSDs 191

7.4. Perspectives and future experiments .................................................................... 192

8. Bibliography ..................................................................................................................... 196

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LIST OF FIGURES

Figure 1.1 Cross correction 29

Figure 1.2 Enzyme delivery via pharmacological ERT and cellular ERT 35

Figure2.1 IgG Enzyme linked immunosorbent assay 67

Figure2.2 Catalytic inhibition assay 68

Figure2.3 Cellular uptake inhibition assay 70

Figure2.4 Preparation of serial dilution of specimens 71

Figure2.5 Cellular uptake inhibition in MPSVI 83

Figure2.6 Western blot analysis 88

Figure3.1 Optimisation of EIA plate coating 93

Figure3.2 Optimisation of ELIZA 94




Figure3.6 Western blot analysis for MPSI 95

Figure3.7 Effect of serum dilutions on catalytic inhibition 97

Figure3.8 Effect of change in pH 98

Figure3.9 Effect of change in dilution buffers 99

Figure3.10 Effect of change in incubation time 100

Figure3.11 Inhibition using two separate incubations 101

Figure3.12 Effect of change in enzyme concentrations 102

Figure3.13 Inhibition of innate enzyme (HEK cells) 103

Figure3.14 Inhibition of innate enzyme (mouse IDUA enzyme) 104

Figure3.15 Optimizing Aldurazyme concentration for uptake inhibition assay 105

Figure3.16 Cellular uptake inhibition at different enzyme concentrations 106

Figure3.17 Percentage cellular uptake inhibition at different enzyme concentrations 107

Figure3.18 Optimizing Myozyme concentration for coating EIA plates 108

Figure3.19 Negative cut off and Sensitivity of ELISA 109

Figure3.20 Negative cut off and Sensitivity of ELISA 109

Figure3.21 Specificity of ELISA assay 110

Figure3.22 Optimizing Myozyme activity assay in 96 well plate 111


Figure3.24 Effect of serum dilutions on catalytic enzyme inhibition 114

Figure3.25 Catalytic inhibition at low enzyme concentrations 116

Figure3.26 Optimizing enzyme concentrations and incubation time for cellular uptake

inhibition assay 117

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Figure3.27 Cellular uptake inhibition at two different enzyme concentrations 118

Figure3.28 Specificity of Cellular uptake assay 119

Figure3.29 Optimizing Naglazyme concentration for EIA plates 120

Figure3.30 Specificity and sensitivity of ELISA 121

Figure3.31 Sensitivity of ELISA 122

Figure3.32 Optimisation of Arylsulphatase enzyme assay in 96 well plate using to different

substrate concentrations 123

Figure3.33 Catalytic inhibition assay using a range of Naglazyme concentrations 124


Figure3.35 Effect of change in serum dilutions 126

Figure3.36 Optimizing Naglazyme concentration for cellular uptake assay 127

Figure3.37 Cellular uptake inhibition assay at different enzyme concentrations and serum

dilutions 128

Figure3.38 Cellular uptake inhibition assay at different enzyme concentrations and serum

dilutions 128

Figure3.39 Optimzing Elaprase concentration for coating EIA plates 129

Figure3.40 Specificity of ELISA and negative cut off 130

Figure3.41 Optimizing iduronate-2-sulphatase enzyme activity assay in 96 well plate 131

Figure3.42 Catalytic inhibition assay 131

Figure3.43 Catalytic inhibition at two enzyme concentrations 132

Figure3.44 Catalytic inhibition at two enzyme concentrations 132

Figure4.1 Immune response (Pre-HSCT) in ERT treated MPSI patients 142

Figure4.2 Immune response (Pre and post HSCT) in ERT treated MPSI patients 145

Figure4.3 Catalytic enzyme inhibition in MPSIH patients 147

Figure4.4 Catalytic enzyme inhibition in MPSIH patients (compared to standard) 147

Figure4.5 Percentage inhibition in tolerized and non-tolerized serum 148

Figure4.6 Cellular uptake inhibition in MPSIH patients 150

Figure4.7 Cellular uptake inhibition in tolerized and non-tolerized MPSIH patients 150

Figure4.8 Correlation between biomarkers (DS/CS ratio) and the antibody titres 152






Figure 5.1 Immune response in long term recipients of ERT (longitudinal data) 163

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Figure 5.2 Percentage catalytic enzyme inhibition compared to a standard (normal serum)

165

Figure 5.3 Percentage catalytic inhibition (direct) in all patients with positive immune

response 165

Figure 5.4 Cellular uptake inhibition in all patients with positive immune response. 166

Figure 5.5 Correlation between ELISA and catalytic inhbition 167

Figure 5.6 Correlation between ELISA and cellular uptake inhbition 168

Figure 6.1 Western Blot analysis for Pompe disease 175

Figure 6.2 Catalytic enzyme inhibition in patients with Pompe disease 176

Figure 6.3 Catalytic enzyme inhibition in patients with Pompe disease 176

Figure 6.4 Specificity of Myozyme catalytic inhibition assay 177

Figure 6.5 Cellular uptake inhibition Pompe disease 178

Figure 6.6 Immune response in patients with MPS VI 180

Figure 6.7 Western blot analysis for Naglazyme 180

Figure 6.8 Catalytic inhibition of enzyme (direct) and against a standard 181

Figure 6.9 Catalytic inhibition of enzyme (direct) and against a standard 181

Figure 6.10 MPS VI Cellular uptake inhibition assay 182

Figure 6.11 MPS VI Cellular uptake inhibition assay 182

Figure 6.12 Correlation between antibody titres and Biomarkers in MPS VI 183

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LIST OF TABLES

Table 1.1 Selected important hydrolases, their natural substrates and Lysosomal Storage

Disorders resulting from their deficiency 23

Table 1.2 Clinical features of LSDs treated with ERT 26

Table 1.3 Indications of HSCT in Lysosomal Storage Disorders 36

Table 1.4 Currently available Enzyme Replacement Therapies (ERT) and incidence of

antibodies in Lysosomal Storage Disorder patients treated with ERT 39

Table 2.1 Fluorescent standard 73

Table 4.1 Patient demographics (longitudinal patient series) 140

Table 4.2 Patient demographics (Cross sectional patient series) 141

Table 4.3 Summary of immune response in longitudinal patient series 143

Table 5.1 MPSI patient demographics (on long term ERT) 160

Table 5.2 Summary of the immune response in MPSI patients (on long term ERT) 162

Table 6.1 Patient demongraphics (Pompe disease) 175

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Abstract

Immune responses in patients with Lysosomal Storage Disorders treated with

Enzyme Replacement Therapy and Haemopoietic Stem Cell Transplantation.

By Muhammad A Saif for the degree of MD, 2012.

The University of Manchester.

Lysosomal storage disorders (LSDs) are caused by defective lysosomal degradation of

macromolecules resulting in accumulation of substrates in various tissues. This gradually leads

to organ dysfunction and the classical clinical presentation with multisystem involvement.

Historically the management of LSDs was confined to symptomatic treatment only. More

recently other therapies have become available. Treatment options include cellular therapy in

the form of Haemopoietic Stem Cell Transplant (HSCT), Enzyme Replacement Therapy (ERT),

Substrate Reduction Therapy (SRT), Chaperone Mediated Therapy (CMT) and gene therapy.

Whilst HSCT and ERT are established strategies in clinical practice for some LSDs, others are

still in the development phase. The easy accessibility of ERT in the developed world (despite a

high cost burden of approximately £144,000 per patient per annum in the UK), fewer risks

associated with its administration and good metabolic and clinical outcome, have made ERT the

treatment of choice for a number of LSDs. In recent years immune response has been identified

as a significant factor in attenuating or nullifying the response to ERT. Despite recognition of this

problem, there is a lack of reliable diagnostic tools to test and evaluate the antibody responses

in the centres delivering ERT and far too little attention has been focused on development,

optimisation and standardization of immune assays.

In this project, IgG ELISA and two different functional enzyme inhibition assays (catalytic

inhibition and cellular uptake inhibition) were developed and optimized. The immune response to

ERT was then studied in recipients of ERT in MPSI, MPSVI and Pome disease. Our practice of

delivering ERT in recipients of allogeneic HSCT prior to transplant provided us with an

opportunity to study the immune response in MPSIH patients during ERT and following HSCT.

We demonstrated functionally active antibodies in long term recipients of ERT in MPSI and

Pompe disease. Allo-immune response in MPSVI did not inhibit the delivered enzyme therapy. A

high titre inhibitory immune response was detected in the majority of MPSIH patients after

exposure to ERT. This immune response was abrogated by allogeneic HSCT rendering these

patients tolerant to replaced enzyme, confirming HSCT as an effective immune tolerance

induction mechanism.

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DECLARATION

I, Muhammad A Saif, declare that no portion of the work referred to in the thesis has

been submitted in support of an application for another degree or qualification of this or

any other university or other institute of learning

COPYRIGHT STATEMENT

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intellectual property (the “Intellectual Property”) and any reproductions of

copyright works in the thesis, for example graphs and tables (“Reproductions”),

which may be described in this thesis, may not be owned by the author and may

be owned by third parties. Such Intellectual Property and Reproductions cannot

and must not be made available for use without the prior written permission of

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ACKNOWLEDGEMENT

I would like to thank my supervisors Dr Rob Wynn and Dr Brian Bigger for their help,

support and guidance throughout this process. I owe my sincere gratitude to my

colleagues in the Stem cell & Neurotherapies laboratory especially Fiona, Alex, Kia, Ai

Yin, Omar, Kenny, Angharad, Karen and Lena. I am greatly indebted to Professor Ed

Wraith, Dr Simon Jones and the medical and laboratory staff at Royal Manchester

Children’s Hospital for their guidance and help.

A special thanks to my wife Anthea and our daughter Ayesha, without their love and

extraordinary patience I would never have been able to complete this work. I am also

grateful to our parents and my sisters who always believed in me.

Above all I am thankful to all the patients, their parents and relatives who participated in

the study and donated blood.

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ABBREVIATIONS

4MU 4-Methylumbelliferyl

ANOVA Analysis of variance

ARSA Arylsulphatase A

BBB Blood brain barrier

BCA Bicinchoninic acid

BEAPs Background enzyme activity in patient serum

BEANs Background enzyme activity in normal serum

CIMPR Calcium independent mannose-6-phosphate receptor

CMT Chaperone mediated therapy

CRIM Cross reactive immunological material

CS Chondroitin sulphate

CSP Clinical surveillance programme

CNS Central nervous system

DS Dermatan sulphate

DMEM Dulbeco’s modified eagle medium

DNA Deoxyribonucleic acid

EAPs Enzyme activity in the presence of patient serum

EANs Enzyme activity in the presence of normal serum

ECL Enhanced chemiluminescence

ELISA Enzyme linked immunosorbent assay

ER Endoplasmic reticulum

14

EIA Enzyme immunoassay

ERT Enzyme replacement therapy

FBS Foetal bovine serum

FVC Forced vital capacity

FDA Food and Drug Administration

GA General anaesthesia

G CSF Granulocyte colony stimulating factor

GAG Glycosaminoglycan

GVHD Graft versus host disease

HSA Human serum albumin

HSCT Haemopoietic stem cell transplant

HPC Haemopoietic precursor cells

HEK Human embryonic kidney cells

HLA Human leucocyte antigens

(h)PGK Human phosphoglycerate kinase

ICD International statistical classification of diseases

IDUA α-L-iduronidase

IDS Iduronate-2-sulphatase

LEBT Lysosomal enzymes from bovine testis

LSDs Lysosomal storage disorder

LV Lentivirus

15

mRNA Messenger ribosomal nucleic acid

MOI Multiplicity of infection

MPS Mucopolysaccharidosis

M6P Mannose-6-phosphate

MPSI H Hurler syndrome

MPSI HS` Hurler Scheie syndrome

MPSI S Scheie syndrome

MLD Metachromatic leukodystrophy

NOEV N-octyl-4-epi-beta-valienamine

OPD o-Phenylenediamine dihydrochloride

OS Overall survival

PAGE Polyacrylamide gel electrophoresis

PDMP 1-phenyl-2-decanoylamino-3-morpholino-1-propanol

RT Room temperature

SRT Substrate reduction therapy

SDS Sodium dodecyl sulfate

TRM Transplant related mortality

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1. Introduction

17

1.1. Lysosomal storage disorders

Lysosomal storage disorders (LSDs) result from defective lysosomal

degradation of macromolecules. This leads to progressive intracellular

accumulation of unhydrolysed substrates. Accumulation of these substances

results in cellular and tissue dysfunction. These disorders are therefore

multisystem, progressive and often cause premature death. However the rate of

progression varies amongst various disease groups and individuals within a

group. LSDs are individually rare but have a combined prevalence of about 1 in

7000 live births worldwide (Meikle, Hopwood et al. 1999; Poorthuis, Wevers et

al. 1999). The majority of these disorders are caused by single gene mutations

in one of the lysosomal enzymes but some include trafficking defects to the

lysosome. Most of these disorders are autosomal recessive but

Mucopolysaccharidosis type II (MPSII), Fabry and Danon disease are X-linked

(Wynn, Stubbs et al. 2009). Over 70 disorders have been described in this

category (Cox and Cachon-Gonzalez 2012). Depending upon a particular

mutation, patients either lack an enzyme or produce an enzyme which does not

function optimally. These mutations have specific clinical outcomes which

explains the heterogeneous phenotype in these illnesses (Vellodi 2005).

Specific mutations have been described for each disease. Whilst there may be a

correlation between genotype and phenotype for some of these disorders, this

relationship is not straightforward and a number of polymorphisms have been

described within the encoding genes which result in different clinical outcomes

for the same genetic mutation. The clinical phenotype is also dependent upon

the level of enzyme secreted. A critical level of lysosomal enzyme is required to

effectively metabolize the substrate within a cell (Gartner, Conzelmann et al.

1983). In an individual with LSD the level of enzyme activity not only determines

the severity of the disease but also the onset of symptoms, treatment outcomes

and immune response to delivered enzyme as outlined in the later sections.

18

1.2. Pathophysiology of LSD

1.2.1. Lysosomes and lysosomal enzymes

Lysosomes are membrane bound organelles which are present in all

mammalian cells except mature erythrocytes. The enzymes (collectively known

as hydrolases) contained in these organelles, hydrolyse a variety of

macromolecules (De Duve, Pressman et al. 1955; De Duve and Wattiaux 1966).

These biochemical reactions take place in a sequential manner in an acidic

environment and result in simpler products of metabolism. About 60 different

hydrolases have been identified so far (Eskelinen, Tanaka et al. 2003). Some

important hydrolases, their substrates and the clinical illnesses arising as a

consequence of their deficiency are described in table 1. The lysosomal

enzymes not only break down the products of cellular metabolism and

extracellular material (endosomes and phagosomes) by formation of secondary

lysosomes (Lloyd 1996; Luzio, Rous et al. 2000) but they are also released

from the cells by a process called exocytosis which can then be taken up by

another cell in proximity.

Synthesis of lysosomal enzymes in the cells is a complex process which takes

place in the rough endoplasmic reticulum. The enzymes then travel to the Golgi

body where they acquire a mannose-6-phosphate (M6P) ligand. Two enzymes,

a phosphotransferase and a diesterase, accomplish this task. This modification

in the structure of the enzyme identifies it as lysosomal. This step is important

because the lack of M6P moiety results in a clinical disorder resembling

mucopolysaccharidosis e.g. Mucolipidoses II and III (Hickman and Neufeld

1972). This step also forms the basis of cross correction; a phenomenon

fundamental to the success of HSCT. During cross correction the enzyme with

M6P ligand is exocytosed by the normal cell (donor) and taken up by an enzyme

deficient cell (recipient) via a M6P or mannose receptor. Cross correction is

19

known to improve clinical outcome in patients receiving HSCT (Hobbs, Hugh-

Jones et al. 1981; Krivit 1983; Krivit, Pierpont et al. 1984).

Despite considerable improvements in the understanding of pathophysiology of

LSDs, a number of questions remain unanswered. The relationship between the

severity of biochemical changes and the mutations that occur in LSDs is still not

clearly understood. A number of genetic abnormalities including deletions,

insertions, duplications and non-sense mutations have relatively straightforward

consequences and lead to a complete lack of protein or dysfunctional protein

structure. However in some cases a small amount of protein is correctly

translated from functional mRNA which leads to an attenuated disease type

(Polten, Fluharty et al. 1991; McInnes, Potier et al. 1992). In contrast, the effects

of genetic abnormalities such as mis-sense mutation and small deletions are

sometimes more difficult to explain based on our current understanding of the

pathophysiology of LSDs. Some of these abnormalities lead to a malfunction of

active site of the enzyme protein (Huie, Hirschhorn et al. 1994; Zhang, Bagshaw

et al. 2000; Garman and Garboczi 2004). This in turn can lead to a significant

compromise in enzyme activity leading to a severe disease phenotype.

However, in some instances, small in-frame mutations and mis-sense mutations

which do not involve the active site, may lead to a severe disease phenotype

(Hermann, Schestag et al. 2000). Some defects lead to misfolding of the protein

structure during their synthesis in the endoplasmic reticulum (ER). This leads to

formation of a protein that cannot be transported out of the ER. Consequently,

these proteins are degraded inside the ER and lead to a severe disease

phenotype (Paw, Moskowitz et al. 1990; Schestag, Yaghootfam et al. 2002;

Poeppel, Habetha et al. 2005). The residual amount of functionally active

enzyme is one of the factors which determine the phenotype of the LSDs. It has

been proposed that alongside genetics, several other factors including

epigenetics and environmental factors determine the disease phenotype. A

D645E missense mutation in the α-glucosidase gene was shown to have an

extremely heterogenous disease phenotype in patients of different ethnic origins

20

(Hermans, de Graaff et al. 1993; Shieh and Lin 1998). Similarly, monozygotic

twins carrying the same gene mutation were reported to have remarkably

different disease phenotypes suggesting a role of environmental factors in

disease pathology (Lachmann, Grant et al. 2004). A disease phenotype

involving one of the most frequent genetic mutations (substitution of asparagine

370 by serine) in Gaucher disease has been studied in great detail. A

consequence of this mutation is the development of a dysfunctional protein

which leads to a base line enzyme level of approximately 10-20% of normal. A

huge variation in the clinical outcome and disease severity was reported in

individuals carrying the same genetic abnormality in this cohort (Beutler,

Nguyen et al. 1993; Zhao and Grabowski 2002).

1.2.2. Cofactors for lysosomal enzymes

Several cofactors are required for the function of lysosomal enzymes.

Deficiency of two important cofactors results in clinically significant disorders.

These cofactors are broadly categorized as “saposins” and “cathepsin like

proteins” which are described below in more detail.

Saposin

Saposins (sphingolipid activator proteins) metabolize glycosphingolipids. Two

genes encode saposins. One gene encodes GM2 gangioliside and the other

encodes prosaposin. Prosaposin is a precursor for saposins A, B, C and D.

Deficiency of these cofactors also results in LSDs. The clinical disorder resulting

from a cofactor deficiency depends upon the enzyme it activates. Unlike most

LSDs, where deficiency of an enzyme would result in one particular disease, the

cofactor deficiency can lead to a more complex clinical phenotype sometimes

resembling more than one clinical entity. Prosaposin, being a precursor

molecule for many cofactors results in more severe illness (Conzelmann and

21

Sandhoff 1978; Christomanou, Chabas et al. 1989; Wenger, DeGala et al. 1989;

Bradova, Smid et al. 1993; Vellodi 2005).

Cathepsin like proteins

A protective protein which has cathepsin A activity, protects the lysosomal B

galactosidase against proteolytic degradation. Lack of this cofactor causes a

combined deficiency of B galactosidase and sialidase (Hoogeveen, Verheijen et

al. 1983; Galjart, Morreau et al. 1991).

1.2.3. Effect of lysosomal enzyme deficiency

Accumulation of unhydrolysed substances in the cells gives rise to structural

and functional changes. In some illnesses the structural changes can be

recognized on light microscopy (The foam cells or sea- blue histiocytes in

Niemann-Pick A, B and C disease and the typical Gaucher cell in Gaucher

disease) but in others ultrastructual analysis is required to visualize the

morphological anomalies (Terry and Weiss 1963; Vanier, Wenger et al. 1988).

Anatomical and physiological stress causes cellular and systemic dysfunction.

This dysfunction can be a consequence of either complex autocrine

dysregulation resulting in altered cellular response e.g. macrophages activation

(Hollak, Evers et al. 1997; Barak, Acker et al. 1999) or simple mass effect due to

the increased size of the cells and tissues (Elleder, Bradova et al. 1990). The

pathogenesis of LSDs is complicated and the phenotypic severity of certain

diseases is not entirely explained by the current understanding of this group of

disorders. The interaction between cytokines/chemokines and macrophages,

neuroinflammation (Wu and Proia 2004), mitochondrial dysfunction (Jolly,

Brown et al. 2002), and intracellular calcium (Lloyd-Evans, Pelled et al. 2003;

Pelled, Lloyd-Evans et al. 2003) play an important role. Some of these factors

have been studied in detail but still the pathogenesis of LSDs remains largely

enigmatic.

22

Lysosomal protein Disease Natural substrate

Galactosidase A Fabry Globotriasylceramide and blood-

group-B

substances

Ceramidase Farber lipogranulomatosis

Ceramide

Glucosidase Gaucher

Glucosylceramide

Sphingomyelinase Niemann–Pick A and B Sphingomyelin

Sphingolipid activator Sphingolipid activator deficiency Glycolipids

Galactosidase GM1 gangliosidosis GM1 ganglioside

Hexosaminidase A GM2 gangliosidosis (Tay–Sachs) GM2 ganglioside and related

glycolipids

GM2-activator protein GM2 gangliosidosis (GM2-activator

deficiency)

GM2 ganglioside and related

glycolipids

Iduronidase MPS I (Hurler, Scheie, Hurler/Scheie)

Dermatan sulphate and heparan

sulphate

Iduronate-2-sulphatase MPS II (Hunter) Dermatan sulphate and heparan

sulphate

Heparan N-sulphatase

(sulphamidase)

MPS IIIA (Sanfilippo)

Heparan sulphate

N-Acetyl-glucosaminidase MPS IIIB (Sanfilippo)

Heparan sulphate

Acetyl-CoA-glucosamide MPS IIIC (Sanfilippo)

Heparan sulphate

N-Acetylglucosamine-6-sulphatase MPS IIID (Sanfilippo) Heparan sulphate

N-Acetylgalactosamine-6-sulphate-

sulphatase

MPSIV (Morquio-A disease) Keratan sulphate, chondroitin-6-

sulphate

Galactosidase MPSIV (Morquio-B disease) Keratan sulphate

N-Acetylgalactosamine-4-

sulphatase

MPS VI (Maroteaux–Lamy) Dermatan sulphate

Beta-Glucuronidase MPS VII (Sly) Heparan sulphate, dermatan sulphate,

chondroitin-4- and -6-sulphates

alpha-Glucosidase Pompe (glycogen-storage-disease type II) Glycogen

Cystinosin Cystinosis Cystine

LAMP2 Danon disease Cytoplasmic debris and glycogen

Sialin Infantile sialic-acid-storage disease and

Salla disease

Sialic acid

Mucolipin-1 Mucoplipidosis (ML) IV Lipids and acid mucopolysaccharides

NPC1 and 2 Niemann–Pick C (NPC) Cholesterol and sphingolipids

23

Cathepsin A Galactosialidosis Sialyloligosaccharides

UDP-N-acetylglucosamine,

N-acetylglucosaminyl-1-

phosphotransferase

Pseudo-Hurler polydystrophy (ML II and ML

III, respectively)

Oligosaccharides,

mucopolysaccharides and lipids

Formylglycine-generating enzyme Multiple sulphatase deficiency Sulphatides

CLN1 (protein

palmitoylthioesterase-1)

Neuronal ceroid lipofuscinosis

(NCL)1 (Batten disease)

Lipidated thioesters

CLN2 (tripeptidyl amino peptidase-

1)

NCL2 (Batten disease) Subunit c of the mitochondrial ATP

synthase

Arginine transporter NCL3 (Batten disease) Subunit c of the mitochondrial ATP

synthase

Table 1.1 Selected important hydrolases, their natural substrates and

Lysosomal Storage Disorders resulting from their deficiency

Various classifications are used for this diverse group of illnesses. The World

Health Organization has given ICD (international statistical classification of

diseases) codes to each of the diseases in this category. This system is not

particularly useful in a clinical setting as many diseases share symptomology.

The most widely used classification is based on the type of substrate

accumulated. Five major groups according to this classification include

Mucopolysaccharidosis, Lipid storage disorders (Sphingolipidosis,

Gangliosidosis and Leukodystrophies), Mucolipidosis, Glycoprotein storage

disorders and Glycogen storage disorders. Some important LSDs are described

in table 1.1.

24

1.3. Clinical manifestations

LSDs can present in infancy, childhood and adult age groups. Infantile

presentations are generally more severe and more frequently present with

neurological symptoms. These are more often rapidly progressive and may be

fatal. Presentation in adults is mostly insidious but it can cause significant

morbidity and disability over a period of time. Juvenile (childhood) forms are

intermediate in severity. These multisystem disorders can involve any organ or

system and frequently affect the central nervous system (CNS), musculoskeletal

(Kovac 2011), reticuloendothelial, cardiovascular and respiratory systems (van

den Berg, de Vries et al. ; Mohan, Hay et al. 2002; Grabowski, Andria et al.

2004; Hoffmann and Mayatepek 2005; Pastores and Meere 2005; Eng, Fletcher

et al. 2007; Yagci, Salor et al. 2009; Damme, Stroobants et al. 2010; Malek,

Chojnowska et al. 2010; Berger, Fagondes et al. 2012; Golda, Jurecka et al.

2012). The neurological symptoms vary from subnormal intelligence to severe

brain stem disorders (Jones, Alroy et al. 1997; Ashworth, Biswas et al. 2006;

Hamano, Hayashi et al. 2008). LSDs can have attenuated phenotypes which

are milder in severity e.g. MPSI has three subtypes known as MPSI H (Hurler

syndrome), MPSI HS (Hurler Scheie syndrome) and MPSI S (Scheie

syndrome). MPSI H is the most severe form and symptoms include progressive

neurodegeneration, visceromegaly, respiratory problems, airway obstruction,

and musculoskeletal abnormalities. This form is diagnosed early (within the first

year of life) and leads to premature death (Cleary and Wraith 1995). Scheie

syndrome (MPSI S) is the mildest form of the disease which is diagnosed much

later (on average at 5 years of age) and has a less severe phenotype. The

symptoms include joint stiffness, facial changes and mild cardio-respiratory

abnormalities. These patients are likely to have a normal life span (Wraith

1995). MPSI HS is intermediate in severity. However, this may result in severe

visceral abnormalities leading to reduced life expectancy. The skeletal

abnormalities in LSDs are quite variable. These changes are sometimes

25

referred to as dysostosis multiplex. Short stature and widespread skeletal

abnormalities are frequent manifestations. Patients with severe vertebral

abnormalities can present with spinal cord compression leading to long term

disability. Organomegaly with hepatic dysfunction and recurrent abdominal pain

can be a presenting feature in some LSDs. Impaired cardiac function is a cause

of serious morbidity in infantile Pompe and can be a limiting factor in delivering

potentially cardiotoxic chemotherapy as a conditioning regimen prior to HSCT

(Kishnani, Hwu et al. 2006; Wynn, Stubbs et al. 2009). Hydrops fetalis has been

reported in some LSDs e.g. Farber’s disease (Kattner, Schafer et al. 1997) and

galactosidosis (Haverkamp, Jacobs et al. 1996). More recently, this was

reported in other LSDs such as MPS IV, MPS VII and NPC (Whybra, Mengel et

al. 2012). Clinical manifestations in patients with LSDs treated with Enzyme

replacement therapy (ERT) are summarized in table 1.2.

26

Type of LSD Clinical manifestations

MPS I

Hurler

Short stature, skeletal abnormalities, hepatosplenomegaly, corneal clouding, cardiac

dysfunction, aiway obstruction, coarse facial features, severe progressive

neurodegeneration, premature death.

Scheie Corneal clouding, cardiac dysfunction, respiratory disease, joint stiffness, normal

intelligence or mild intellectual impairment.

Hurler-Scheie

Mild cognitive impairment, skeletal abnormalities, hepatosplenomegaly, respiratory

disease, cardiac dysfunction, corneal clouding (severity of clinical phenotype is usually

intermediate between Hurler and Scheie).

MPS II ( Hunter)

Variable in severity. Skeletal abnormalities (short stature, coarse facial features),

hepatosplenomegaly, macroglossia, progressive neurodegeneration (variable severity),

respiratory disease, airway obstruction, cardiomyopathy, recurrent infections.

MPSVI (Maroteaux-Lamy) Short stature, coarse facial features, skeletal abnormalities, joint stiffness, organomegaly,

corneal clouding.

Pompe

Infantile form: hypotonia, failure to thrive, respiratory failure,cardiac failure,

hepatosplenomegaly, macrglossia

Adult form: skeletal muscle abnormalities, respiratory failure (insidious), cardiac

dysfunction

Gaucher Hepatosplenomegaly, pancytopenia, liver cirrhosis, osteoporosis, skeletal abnormalities,

neurological involvement (convulsion, dementia, muscle apraxia) in type II and III only.

Fabry Renal failure, cardiac dysfunction, cardiomyopathy, cataract, optic atrophy, Small fibre

neuropathy, vasculitis,

Table 1.2 Clinical features of LSDs treated with ERT (Cox and Schofield

1997; Brady and Schiffmann 2000; van der Beek, Hagemans et al. 2006)

27

1.4. Treatment strategies

1.4.1. Haemopoietic stem cell transplant (HSCT)

History

A zygote (totipotent stem cell) is the first cell of a new individual. This cell

divides to form pluripotent stem cells. Pluripotent stem cells retain the ability to

differentiate into any cell derived from the three germinal layers. Multipotent

stem cells are more committed cells derived from pluripotent precursors. These

can further differentiate into a closely related family of cells. Multipotent

haemopoietic stem cells are critical for HSCT as they are capable of

reconstituting the entire lympho-haemopoietic system. The first stem cell

transplants were done over 50 years ago in patients with leukemia and

immunodeficiency. In 1959, Thomas reported the first bone marrow transplant in

a patient with end stage leukemia. The source of stem cell was the bone

marrow of an identical twin. This patient survived nearly three months (Thomas,

Lochte et al. 1959).

The first HSCT for LSDs was reported in a patient with MPSIH in 1981 (Hobbs

1981). The rationale for transplantation in LSDs, is to deliver cellular enzyme

replacement therapy from engrafted donor blood cells. Haemopoietic precursor

cells (HPC) progeny differentiate into tissue macrophages including Kupfer cells

in liver and microglial cells within the brain parenchyma of the recipient.

Lysosomal enzymes secreted from these normal donor cells are taken up by the

enzyme deficient recipient cells in the vicinity, via the Mannose or M6P receptor.

This phenomenon is known as cross-correction (Neufeld 1983) and results in

biochemical and phenotypic recovery of recipient cells (Hobbs, Hugh-Jones et

al. 1981; Krivit 1983; Krivit, Pierpont et al. 1984) (see figure 1.1).

In 1960, Human leucocyte antigens (HLA) were identified and it became

possible to match the HLA types of the donors and recipients. This paved the

way for further development of HSCT as a viable treatment option which has

evolved considerably since then. Various types of conditioning regimens and

28

stem cell sources are now in use. HLA matching and immunosuppression is

more sophisticated and effective. All these changes have resulted in steady

improvement in the outcome of HSCT over the years.

Principles of HSCT

HLA matching

The process of HSCT involves carefully selecting the donor and source of stem

cells. A sibling has a 1/4 chance of being a match for the patient. However,

transplantation in genetic disorders, means that 2/3 of the matched siblings are

likely to be heterozygote carriers of the disease. This observation is important

becuase the carriers of defective genes may deliver a reduced amount of

enzymes to their affected siblings. The patient is typed for HLA class I (A, B and

C) and class II (DR and DQ). For unrelated donor transplantation from

peripheral blood or bone marrow sources the donor is matched for all 10 loci. It

is not always possible to find a 10/10 match. Greater disparity between the

donor’s HLA type and the recipient’s can increase transplant complications e.g.

Graft versus host disease and transplant related mortality (TRM) (Diaconescu,

Flowers et al. 2004; Sorror, Maris et al. 2004; Copelan, Casper et al. 2007).

Umbilical cord blood, on the other hand requires less stringent matching. It is

sufficient to match the cord blood at 6 loci instead of 10 (HLA A, B and DR).

Many centres use up to a 4/6 match for cord blood transplant in genetic disease.

29

Figure1.1 Cross-correction

Innate enzymes are glycosylated (green circles) in the endoplasmic reticulum

(ER) of normal cells. Further modification to inocorporate the mannose 6-

phosphate takes place in the Golgi apparatus (Golgi) where they bind the

mannose 6-phosphate receptor (M6PR). The majority (heavy arrows) of the

enzymes are then transported to the lysosome (Lys) whilst a minority (fine

arrows) are secreted from the cell. The secreted enzyme can bind the cell

membrane M6PR or the mannose receptor (ManR), respectively and are

internalized. M6PR is universally expressed on all cells, whereas ManR

expression is limited to cells of the reticuloendothelial system (Modified from

(Sands and Davidson 2006))

30

Sources of stem cells

The following three sources of stem cells are currently in use for HSCT.

Bone marrow (BM)

Bone marrow is usually taken from the iliac crest by bone marrow needles. This

requires general anaesthesia (GA) and multiple needle insertions into the pelvic

bone to aspirate the bone marrow. The incidence of graft versus host disease

(GVHD) with bone marrow stem cells is lower but the cell engraftment may take

longer when compared to peripheral blood as a source of stem cells.

Peripheral blood (PB)

Stem cells are mobilized from the bone marrow into the peripheral blood with

the help of granulocyte colony stimulating factor (G-CSF). The mobilized stem

cells are then collected by leukapheresis. This does not require GA. A

peripheral blood stem cell transplant (PBSCT) tends to engraft quicker than the

bone marrow but the incidence of graft versus host disease (GVHD) may be

higher (Schrezenmeier, Passweg et al. 2007).

Umbilical cord blood (UCB)

Fetal blood is rich in stem cells. After birth the fetal blood is collected from the

placenta. The umbilical cord blood is a suitable source of transplant because it

is rapidly available, poses no risk to the donor, contains less risks of disease

transmission, and requires less precise matching. The dose of stem cells in cord

blood collection is generally lower than peripheral blood and bone marrow, and

the engraftment and immune reconstitution can be slower. Recent data has

shown that the enzyme delivery, donor chimerism and outcome of HSCT is

superior when cord blood is used as a source of stem cells, hence it is has

become a preferred method for LSD patients receiving HSCT in many centres

(Staba, Escolar et al. 2004; Prasad and Kurtzberg 2008; Prasad, Mendizabal et

al. 2008).

31

Conditioning

Chemotherapy and radiotherapy are used to prepare the recipients for

transplantation. This is called conditioning. In children, receiving HSCT for LSD,

the conditioning almost always involves chemotherapy without the use of

radiotherapy. The purpose of conditioning is to empty the bone marrow niche so

that the stem cells from the donor can be engrafted in that space. It also

weakens the recipient immune system which facilitates the engraftment of the

donor cells. In malignancies, conditioning reduces the disease burden prior to

transplant. Various types of conditioning regimens are in use. Broadly the two

main types of conditioning are myeloablative and non-myeloablative or reduced

intensity. Myeloablative conditioning used for HSCT in LSDs is usually based on

busulfan conditioning. Cyclophosphamide and/ or Melphlan is used in

combination with busulphan. Use of T cell depletion (Campath or Antithymocyte

globulin) depends upon the donor type and the risk of GVHD. A number of non-

myeloablative conditioning regimens are in use in different transplant centres

and may include non-myeloablative doses of chemotherapeutic agents including

treosulphan, cyclophosphamide, melphalan etc. Use of chemotherapy inevitably

contributes to the risks of HSCT. Major side effects of chemotherapy are organ

toxicity, multiorgan failure, myelosuppression, immunosuppression, infertility

and risk of secondary malignancies (Shalet, Didi et al. 1995; Michel, Socie et al.

1997; Cohen, Bekassy et al. 2008; Faraci, Bekassy et al. 2008; Friedman, Rovo

et al. 2008; Tichelli, Passweg et al. 2008; Tichelli, Rovo et al. 2008).

At the end of conditioning the stem cells are infused to the patient. A period of

myelosuppression and immune suppression follows the conditioning

chemotherapy. During this phase immunosuppressive therapy is continued to

prevent GVHD. Prophylactic antimicrobials are given for a prolonged period of

time to prevent opportunistic infections. The short term risks of transplant are

mainly due to the toxicity of conditioning, myelosuppression,

immunosuppression, acute GVHD and multi organ failure requiring intensive

care support. These contribute to the high transplant related mortality (TRM)

32

immediately after the transplant (Hayes, Lush et al. 1998). HSCT may be

associated with long term side effects and significant medium to long term

morbidities. These include short stature (Polgreen, Tolar et al. 2008),

endocrinopathies (Ho, Lewis et al. 2011), premature cataract, secondary

cancers (Niethammer and Mayer 1998) and organ dysfunction due to chronic

GVHD. In summary the risks of transplant may be due to the toxicity of

chemotherapeutic agents used during conditioning, myelosuppression,

immunosuppression and graft versus host disease. In every patient, under

consideration for HSCT, the risks and benefits must be weighed carefully.

The outcome of HSCT in lysosomal diseases

The first allogeneic HSCT for LSD in a patient with MPS IH resulted in normal

development and intelligence (Boelens, Prasad et al. 2010). Over the last three

decades nearly 2000 patients have been transplanted for LSDs in Europe and

North America. Early experience of HSCT in MPS I showed that it led to

improvements in cardio-respiratory symptoms, ocular changes, organomegaly

and psychomotor symptoms (Whitley, Ramsay et al. 1986; Summers, Purple et

al. 1989; Braunlin, Hunter et al. 1992; Haddad, Jones et al. 1997; Vellodi, Young

et al. 1997). HSCT in MPSVI was shown to improve clinical outcome and

neurocognition in the recipient (Krivit, Pierpont et al. 1984). Similarly,

encouraging outcomes were reported after HSCT in late onset metachromatic

leukodystrophy (MLD) (Imaizumi, Gushi et al. 1994). So far, HSCT has been

undertaken in a number of LSDs including MPS II, MPS III, MPS VI, α-

Mannosidosis, globoid cell leukodystrophy with variable clinical outcome (Krivit,

Pierpont et al. 1984; Vellodi, Young et al. 1992; Coppa, Gabrielli et al. 1995;

Wall, Grange et al. 1998; Kapaun, Dittmann et al. 1999; Krivit, Aubourg et al.

1999; Turbeville, Nicely et al. 2011). Current experience of the use of HSCT in

LSD is summarized in table 1.3.

In 2008, Moore et al. published data on long term survival of MPS I patients in

the UK. The survival of the patients who received HSCT was superior to those

who did not receive transplant. The probability of survival after BMT was 68%,

33

66% and 64% at two, five and ten years (Moore, Connock et al. 2008). Over the

last two decades the overall survival (OS) of HSCT in MPS I patients has been

reported to be around 40-70% (Peters, Balthazor et al. 1996; Peters, Shapiro et

al. 1998; Souillet, Guffon et al. 2003). Data from Eurocord and Duke University

showed that the OS in the patients transplanted after 2004 was 75% (and above

80% in registry reported grafts for the most recently transplanted children) and

before 1999 it was 53%. In some centres this figure is now approaching 90%

(Boelens, Rocha et al. 2009; Wynn, Mercer et al. 2009). The improvement in the

overall survival has been attributed to the changes in the clinical practice of

HSCT (Vellodi, Young et al. 1997; Grewal, Krivit et al. 2002). A multicentre

study looked at the risk factors for graft failure in 2007 and identified three major

reasons for improved survival in these patients. Firstly, an effective conditioning

regimen was developed which included intravenous busulfan with

pharmacokinetic monitoring. Secondly, use of full intensity conditioning

regimens which resulted in better engraftment. Thirdly, use of a T cell replete

graft which reduced graft rejection resulting in improved outcome (Boelens,

Wynn et al. 2007). This group also showed that the metabolic outcome of

unrelated donor transplant recipients was superior to those who received ERT

alone (Wynn, Wraith et al. 2009). In this study, urinary dermatan suplphate

(DS) to chondroiton sulphate (CS) ratio was measured to evaluate metabolic

recovery in the patients receiving ERT and this was compared to the recipients

of sibling and unrelated donor HSCT. This study showed that unrelated donor

transplant most effectively reduced the residual substrate, followed by

heterozygous donor transplants (sibling). Substrate reduction was lowest in the

recipients of ERT. Even though the enzyme levels and immune response in

ERT treated patients were not measured, it is possible that the inferior outcome

in this group of patients was due to the reduced level of functional enzyme

which was neutralized by inhibitory antibodies. This is supported by a close

analysis of the data, in this study, which reveals that some patients showed

significantly worse biomarker response when compared to the other ERT

recipients (Wynn, Wraith et al. 2009).

34

For an individual patient undergoing HSCT a number of factors determine the

outcome after transplant (Wynn 2011) which include type of disease,

genotype/phenotypic characteristics, age at the time of transplant, use of ERT

prior to HSCT (Tolar, Grewal et al. 2008), availability and efficacy of alternative

treatments, complications of applied therapy and a multidisciplinary approach.

Carefully balancing these factors is likely to result in improved outcome of HSCT

in LSDs. A number of studies have consistently shown that the HSCT improves

the clinical outcome including organomegaly, functional and neurophysiological

outcomes, particularly if done earlier in asymptomatic patients (Peters,

Balthazor et al. 1996; Vellodi, Young et al. 1997; Guffon, Souillet et al. 1998;

Peters, Shapiro et al. 1998; Staba, Escolar et al. 2004).

In neurocognitive disorders, HSCT is superior to ERT due to its ability to correct

CNS disease by donor derived mononuclear cells. These cells interact with the

adhesion molecules on the surface of the brain capillary endothelium and result

in transmigration of cells into the brain parenchyma (Martin-Padura, Lostaglio et

al. 1998). This complex process starts with adhesion of leucocytes to

endothelial receptors followed by transient opening of endothelial junction to

allow migration of the cells through the endothelium (Martin-Padura, Lostaglio et

al. 1998). These cells subsequently engraft within the brain tissue and become

resident macrophages known as microglial cells, providing deficient enzyme to

affected cells by cross correction.

35

Figure 1.2 Enzyme delivery via pharmacological ERT and cellular ERT

Pharmacological ERT (infused) stays in the circulation and is delivered to the peripheral tissues. However, this ERT cannot penetrate the blood brain barrier and hence fails to correct the CNS disease. In contrast, cellular ERT (allogeneic HSCT) delivers enzyme into the CNS via donor derived microglial cells (Wynn 2011).

36

Indication Disease

Standard therapy

MPSIH (Hurler)

MPSVII (Sly)

alpha-mannosidosis

Pre-symptomatic Krabbe

Late onset globoid cell leukodystrophy

Wolman

Optional

MPSIH/S

MPSIS

MPSVI

MPSVII

Late onset Krabbe

Fucosidosis

Farber

Gaucher (Non neuronopathic and norrbottnian)

Niemann-pick C

Investigational

MPSIIA

MPSIIB

MPSIII A

MPSIIIB

MPSIIIC

Unknown

Gaucher (acute neuronopathic and subacute

neuronopathic)

Tay sachs (early onset)

MLD (early onset)

Sandhoff

Niemann-pick A

Niemann-pick B

Table 1.3 Indications of HSCT in Lysosomal Storage Disorders

(These classifications were written as a guide to the commissioners of transplant services in the UK)

37

1.4.2. Enzyme replacement therapy (ERT)

The basic concept of enzyme replacement therapy was first described by de

Duve in 1964 (Deduve 1964). The initial attempts to treat LSDs by enzyme

replacement therapy were made in the 1970s. A number of clinical studies were

undertaken to see the effects of intravenous infusion of deficient enzymes in

Pompe, Fabry and type I Gaucher disease. Human urine or cord blood was

used as a source to extract lysosomal human enzyme. Unfortunately this

enzyme treatment failed to achieve therapeutic levels of deficient enzymes and

hence was abandoned. Around that time HSCT was introduced as a therapy for

LSDs showing promising results in some diseases in this group and resulting in

further decline in interest in ERT. In the 1980’s, attempts were made to prepare

more purified forms of lysosomal enzyme. Genzyme purified Beta glucosidase

from human placenta and manufactured it on an industrial scale. This treatment

proved very successful with excellent haematological, biochemical and clinical

response in patients and radically changed the management of Gaucher

disease (Barton, Furbish et al. 1990; Barton, Brady et al. 1991). The original

product (Ceredase) was subsequently replaced by recombinant human enzyme

produced in Chinese hamster ovary cells (Cerezyme) (Wraith 2006). Following

the commercial success of recombinant human glucocerebrosidase a great deal

of interest was generated in this potentially lucrative area. This encouraged the

development of new recombinant human enzyme therapies for the treatment of

other LSDs. During this treatment patients receive regular intravenous infusions.

This is essentially a lifelong therapy to replace the deficient enzyme and may

require insertion of long term tunneled intravenous catheters. After infusion into

the veins the administered enzyme is taken up by the deficient cells by

endocytosis. As explained earlier, delivery of the enzyme to the lysosomes

within a cell is dependent upon mannose or M6P receptors. Therefore it is

crucial that the delivered recombinant human enzyme is recognized by the

appropriate receptor. This is achieved by modification of the glycoprotein

structure during the manufacture of recombinant human enzyme (Richards).

38

Since the development of alglucerase (Cerezyme) for type 1 Gaucher Disease

(1991), a number of other LSDs have been treated with ERT. This treatment

however cannot correct the CNS disease due to its inability to penetrate through

the blood brain barrier (BBB). This limitation is discussed in detail in a later

section. Currently available ERTs are shown in the table 1.4.

39

Disease Defective

enzyme

Substrate

accumulation

Enzyme

replacement

therapy

Incidence

of IgG

antibody

(%)

Gaucher Disease

Acid beta glucosidase

(glucocerebrosidase)

Glucosylceramide

Alglucerase

(Ceredase)

12.8

Imiglucerase

(Cerezyme)

13.8

Fabry Disease Alpha galactosidase

A

Globotriaosylceramide Agalsidase beta

(Fabrazyme)

Agalsidase alfa

(Replagal)

90

Hurler Syndrome

(MPSI)

Alpha L-iduronidase dermatan and

heparan

sulfates

Iduronidase

(Aldurazyme)

91

Pompe Disease

(GSD II)

Acid alpha

glucosidase

Glycogen Myozyme 89

Hunter’s

(MPSII)

Iduronate-2-sulfatase Dermatan and heparin

sulfates

Elaprase 51

Niemann-Pick B

Disease

Sphingomyelinase

NPC1 and 2

Cholesterol and

sphingolipids

Recombinant human

acid sphingomyelinase

(rhASM)

NA

Maroteaux-Lamy

(MPS VI)

N-

acetylgalactosamine-

4-sulfatase

Dermatan sulphate Naglazyme

arylsulfatase B

97

Table 1.4 Currently available Enzyme Replacement Therapies (ERT)

and incidence of antibodies in Lysosomal Storage Disorder patients

treated with ERT.

40

The outcome of ERT

An ideal ERT should be able to deliver therapeutic enzyme levels, have minimal

side effects associated with its administration, stop the disease progression and

correct the existing disease manifestations in patients. The outcome of ERT in

Gaucher disease and MPSI has probably been scrutinized more than any other

LSD. In 2002, Weinreb et al published 2-5 year follow up data on 128 patients

with Gaucher disease. Patients on ERT experienced significant improvement in

various parameters evaluated during this study including haematological

indices, hepatomegaly (30-40% reduction), splenomegaly (50-60%) and disease

related episodes of pain (half of the patients were pain free within two years).

Recombinant human acid beta glucosidase is now the standard treatment for

patients with Gaucher disease (Weinreb, Charrow et al. 2002). Similar results

were reported in a phase I study in MPSI patients who received ERT

(recombinant human alpha-L-Iduronidase- laronidase). In an initial study, 12

patients with MPSI (age 5-22 years) had clinical radiological and biochemical

assessment on week 6, 12, 26 and 56 after commencing the treatment.

Radiological investigations included magnetic resonance imaging of the brain

and abdomen and echocardiogram of the heart. A significant improvement in

clinical outcome was described. Hepatosplenomegaly improved in all patients

(liver size in 8 patients was reported to be within normal range by six months).

Cardiac dysfunction ameliorated in all patients and ejection fraction improved on

echocardiogram (Kakkis, Muenzer et al. 2001). Subsequently a double blind

placebo controlled trial compared the clinical outcome of 22 patients (18 MPSI

HS and 4 MPS IS) treated with laronidase with that of 23 patients who received

placebo. An improvement in visceromegaly, forced vital capacity (FVC) and 6

minute walk test was reported in laronidase group (Wraith, Clarke et al. 2004).

This study, which was originally designed for 26 weeks, was extended into an

open label clinical trial for a period of 72 weeks and confirmed ongoing clinical

response in the patients treated with laronidase (Wraith 2005).

41

To evaluate the safety and efficacy of recombinant human iduronate-2-sulfatase

(idursulfase) in the treatment of MPS II, 96 patients were enrolled in a double-

blind, placebo-controlled trial. At the time of evaluation at one year, patients in

ERT group showed statistically significant improvements in clinical parameters

compared to the placebo group (Muenzer, Gucsavas-Calikoglu et al. 2007). In

another randomised control study in MPS II patients (n=12), idursulfase group

had lower urinary GAG and showed an improvement in hepatosplenomegaly

and exercise tolerance (Muenzer, Wraith et al. 2006). Several other studies

subsequently confirmed the efficacy of ERT in MPS II (Giugliani, Federhen et al.

2010; Schulze-Frenking, Jones et al. 2010).

A number of phase I-III trials of arylsulfatase B (recombinant human N-

acetylgalactosamine 4-sulfatase) for MPS VI, confirmed the superior clinical and

biochemical outcome of patients treated with ERT (Harmatz, Giugliani et al.

2006; Harmatz, Yu et al. 2010). Recently the European clinical surveillance

prgramme (CSP) published an initial report of an intended 15 yearlong study

which showed significant ongoing improvement in organomegaly and clinical

outcome in patients on ERT (Hendriksz, Giugliani et al. 2011). Several other

reports suggested improved outcome in ERT treated MPS VI patients (Decker,

Yu et al. 2010; Giugliani, Federhen et al. 2010; Lin, Chen et al. 2010).

Results of two clinical trials were published for Fabry disease in 2001. Both

agalsidase alfa (Schiffmann, Kopp et al. 2001) and agalsidase beta (Eng,

Banikazemi et al. 2001; Eng, Guffon et al. 2001) showed promising results in

phase I-III trials and improved clinical and biochemical parameters. Thurberg et

al. also reported reversal of histological changes in Fabry disease patients

treated with agalsidase beta (Thurberg, Randolph Byers et al. 2004). The

enzyme replacement therapy also improved symptoms in female carriers of the

disease who had some disease manifestations (Baehner, Kampmann et al.

2003).

Initial reports of recombinant human alpha-glucosidase as a treatment in Pompe

disease patients were encouraging and confirmed its safety (Van den Hout,

42

Reuser et al. 2001). Winkell et al. reported three wheelchair-bound patients who

showed signs of disease stabilization and some recovery in skeletal muscle

function (Winkel, Van den Hout et al. 2004). An open-label study of ERT in 44

late-onset Pompe disease patients showed improvement in 6-min walk tests

and serum creatinine kinase levels (Strothotte, Strigl-Pill et al. 2010). A UK

survey of 20 infantile Pompe disease patients treated from 2000 to 2009 with a

median duration of treatment of 31 months showed an overall survival of 65%

and ventilator-free survival of 35% (Chakrapani, Vellodi et al. 2010). However, in

this study, 35% (7/20) patients were reported to have died at a median age of

10 months which was worse than the original clinical trials on the recombinant

enzyme. This was attributed to the late diagnosis and a higher number of

infantile Pompe disease patients with a more severe clinical illness.

Improvement in the clinical and biochemical parameters in Pompe disease has

also been reported in other publications(Van den Hout, Reuser et al. 2001; Van

den Hout, Kamphoven et al. 2004; Chakrapani, Vellodi et al. 2010; Strothotte,

Strigl-Pill et al. 2010).

1.4.3. Substrate reduction therapy (SRT) and chaperone mediated

therapy (CMT)

As discussed earlier, ERT and HSCT work by delivering the enzyme to the

enzyme deficient individual. This in turn reduces the substrate accumulation in

the body by metabolizing the substrate. In contrast SRT works by slowing down

the accumulation of substrate in the cells or driving substrates down alternative

degradation pathways. Platt et al. in 1994 described an N-alkylated imino-sugar

compound, N-butyl-deoxynojirimycin (Miglustat), to be a potent inhibitor of the

ceramide-specific glucosyltransferase. This substance inhibited synthesis of

glycosphingolipids and prevented accumulation of glucosylceramide in an invitro

model of Gaucher disease. Radin and colleagues in 1996 proposed that it

would be feasible to slow the synthesis of glucosylceramide by an inhibitor drug,

43

1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), to attain clinical

and biochemical recovery in Gaucher disease (Radin 1996). In 2000 Cox et al

published data from a human trial in Gaucher disease patients receiving oral

Miglustat. This study showed modest clinical and haematological improvement

(Cox, Lachmann et al. 2000). Miglustat (Zavesca) was approved for use in type I

Gaucher disease by European Committee for Proprietary Medicinal Products

(CPMP) in July 2002 and subsequently the Food and Drug Administration (FDA)

granted approval for its use in USA in July 2003 (Pastores 2006). The results of

miglustat phase I and II studies in Niemann-Pick type C (Pineda ; Pineda,

Wraith et al. 2009) and chronic GM2 gangliosidosis type Sandhoff (Masciullo,

Santoro et al.) show modest responses, but this treatment is not curative for any

of these LSDs.

In recent years, there has been an increasing interest in a naturally occurring

tyrosine kinase inhibitor (genistein) which can cross the BBB. It reduced the liver

lysosomal compartment size in MPSIIIB mouse model after eight weeks of

treatment (Malinowska, Wilkinson et al. 2009). In a recent study involving 9

months of continuous treatment with genistein in the same mouse model,

significant improvement in lysosomal storage, substrate reduction and

neuroinflammation were noted. These changes translated into correction of

behaviour in the mice (Malinowska, Wilkinson et al. 2010).

Chaperones are small molecular weight ligands which bind to mutated

lysosomal proteins and stabilize them. This results in functional activation of the

defected proteins. Chaperone mediated therapies (CMT) e.g. imino acids (N-

butyldeoxynojirimycin) and N-octyl-4-epi-beta-valienamine (NOEV) have been

tested in glycosphingolipidoses (Priestman, Platt et al. 2000; Butters, Dwek et

al. 2005) and beta-galactosidosis (Matsuda, Suzuki et al. 2003) respectively. In

a recent study, 1-deoxynojirimycin derivatives were shown to be more potent

inhibitors of D-galactosidases. This has resulted in renewed interest in studying

their efficacy in the treatment of GM1-gangliosidosis (Frohlich, Furneaux et al.

2011).

44

These therapies are inexpensive, easy to administer, generally well tolerated

and have a promising side effect profile. However, limited clinical efficacy

remains the main limitation of these treatments. Although neither SRT nor CMT

is curative as a single agent therapy, their utility as combination or adjuvant

therapies in milder disease phenotypes is yet to be assessed.

1.4.4. Gene therapy

Gene therapy aims to replace a mutated gene with functional copies of a normal

gene to correct the phenotype of the disease (Ponder and Haskins 2007). Once

incorporated into the cellular DNA, the gene is transcribed and translated to

provide a permanent source of enzyme which can correct the disease

phenotype (Langford-Smith, Wilkinson et al. 2012). Gene therapy can be

delivered by several techniques which mostly utilize a delivery mechanism

known as a vector. However, it is possible to deliver the gene directly and

achieve a limited local response (Sikes, O'Malley et al. 1994; Yoo, Soker et al.

1999). A major disadvantage of this approach is rapid degradation of gene

product in vivo. A number of vectors have been used to deliver stable copies of

gene into the cells. Vectors can be broadly classified as viral vectors and non-

viral vectors. Adeno-Associated viral vector (Koeberl, Alexander et al. 1997;

Alba, Bosch et al. 2005; Haisma and Bellu 2011), retroviral vectors (Dani 1999)

and lentiviral vectors (Zufferey, Dull et al. 1998) have been used in gene therapy

approaches. Non-viral vectors involve delivery of gene via chemical or physical

carrier(Niidome and Huang 2002). HSCT can be used to deliver gene therapy

(Emery, Nishino et al. 2002). Due to the improvement in outcome of HSCT,

gene delivery by HSC using a viral vector such as lentivirus (LV-HSCT) has

become feasible. This approach has been used in animal models of several

neurodegenerative LSDs. LV-HSCT has shown promising results in

metachromatic leukodystrophy (Biffi, Capotondo et al. 2006), MPSI (Visigalli,

Delai et al. 2010) and MPS IIIA (Langford-Smith, Wilkinson et al. 2012). Even

45

though gene therapy is still a research tool, based on the recent data, a clinical

trial is underway to evaluate the efficacy of gene therapy in MLD.

1.5. Limitations of treatments

The limitations of the two main treatments used in clinical practice i.e. HSCT

and ERT are discussed in detail in following sections.

1.5.1. Limitations of HSCT

As mentioned earlier, HSCT is now a standard treatment for some LSDs e.g.

MPSIH. However, even in this disorder the clinical outcome is inconsistent in

different tissues. It is less effective in prevention and reversal of some disease

manifestations e.g. musculoskeletal and orthopaedic problems (Masterson,

Murphy et al. 1996; Weisstein, Delgado et al. 2004). Early reports of HSCT in

several LSDs including Hunter syndrome (Shapiro, Lockman et al. 1995;

McKinnis, Sulzbacher et al. 1996) and Gaucher (Rappeport and Ginns 1984;

Tsai, Lipton et al. 1992) showed some efficacy but relatively high morbidity and

mortality. HSCT is ineffective in other LSDs where the results of transplant are

not encouraging e.g. the initial reports of HSCT in patients with MPSIII

(Sivakumur and Wraith 1999) showed that the HSCT did not prevent

neurological deterioration even if the transplant was performed early in life.

More recently a better outcome was reported in 19 patients with MPSIII in a

single centre study. 12 patients survived the transplant and 9 of them were

reported to have some degree of disease stabilization. In this cohort most of the

patients were transplanted late in life and only two patients received HSCT

before the age of 2 years. Even in the patients who were transplanted late

during their life (3-5 years), some improvement was reported in cognitive skills.

In this cohort the clinical outcome was assessed by various clinical and

neurophysiological parameters and was found to be better than the non-

transplanted patients. However in comparison to the normal controls a

generalized developmental delay was reported. Despite these initial

46

encouraging results, it is too early to conclude about the long term outcome of

transplant in these patients (Prasad, Mendizabal et al. 2008). The reason for

poor outcome in some LSDs is not entirely clear. In animal models of MPSIII,

the transplant reduced the heparan sulphate derived disaccharides by 27% in

brain parenchyma but the secondary cholesterol and GM3 ganglioside storage

was not significantly altered and hence did not translate into clinical

improvement (Lau, Hannouche et al. 2010). This transmigration does not deliver

therapeutic enzyme levels in the brain and fails to achieve neurological recovery

by cross correction. It has been proposed that the delivery of enzyme into the

tissue could be improved by either increasing the enzyme available to the tissue

or improving the tissue specific transport coefficient (Wynn et al 2009). Graft

manipulation to achieve higher gene expression could deliver

supraphysiological levels of enzyme to the brain tissue. This might offer an

effective cellular therapy in these patients in future.

In summary HSCT is a promising treatment for some LSDs but it has some

limitations. It is not effective in all LSDs. Even though the outcome of HSCT has

been steadily improving, it still carries significant morbidity and mortality. Apart

from short term risks of relatively high transplant related mortality (TRM), the

later complications of transplant, including risk of secondary malignancies and

infertility, need to be considered prior to contemplating an HSCT. Also, this

therapy does not improve the clinical phenotype completely in some patients,

particularly if offered late in life. It does not alter the clinical course of illness in

some organs e.g. musculoskeletal system. The maximum effect of treatment is

achieved if given early (with in the first two years) which is not always possible

due to late presentation in some LSDs or delay in diagnosis due to a lack of

awareness of these rare disorders amongst the wider medical community.

47

1.5.2. Limitations of ERT

Even though ERT improved outcome and reversed some clinical symptoms, it

failed to show universal benefits in halting and reversing the disease

progression and clinical course of disease (Mercimek-Mahmutoglu, Reilly et al.

2009). There is increasing evidence from animal studies and human trials to

suggest that the ERT is less effective in reversing disease pathology in some

organs and clinical recovery in other systems can take a long time. Bone

disease prior to ERT is not corrected during the treatment and some bone

pathologies e.g. osteopenia and osteoporosis may take over 5 years to resolve.

ERT is less effective in slowing and reversing the disease pathology in bone,

cartilage, musculoskeletal system, heart valves and brain (Schiffmann, Kopp et

al. 2001; Wraith 2006; Cox-Brinkman, van den Bergh Weerman et al. 2010). In

a follow up study of MPSI patients which lasted 6 years, clinical improvement

was slow and took several years in some patients, despite relatively quick

recovery in urinary GAG and enzyme levels (Sifuentes, Doroshow et al. 2007).

A major problem with the use of ERT is its inability to correct the CNS disease.

This is because the blood brain barrier (BBB) prevents the delivered therapy

from penetrating into the brain tissue (Begley, Pontikis et al. 2008; Enns and

Huhn 2008). BBB is meant to protect the brain tissue from potentially harmful

substances and microorganisms present within the circulation. This barrier is

formed by tight endothelial junctions and astrocyte projections (glia limitans)

surrounding the brain capillaries. This mechanical and electrical barrier prevents

most solutes including ERT in peripheral circulation from entering the brain

parenchyma freely (Begley and Brightman 2003). In order to effectively treat

neurological involvement in LSD the treatment should be able to cross the BBB

either by disruption of the barrier or by direct infusion of the ERT into the brain.

Recently ERT was successfully delivered into cerebrospinal fluid via

cerebellomedullary cistern in animal models of MPSIIIB(Jolly, Marshall et al.

2010). The success of this approach has led to the initiation of a clinical trial in

MPSIIIA patients via the intrathecal route.

48

As discussed earlier, the cost of treatment is an important consideration in a

climate where resources are becoming increasingly scarce. ERT carries a

relatively high cost burden estimated at $150,000-300,000 per patient per

annum in MPS I (Connock, Juarez-Garcia et al. 2006) and for all ERT treatable

LSDs, averages £144,000 per patient in the UK. This makes it almost

impossible to administer this treatment in patients in the developing world. Even

in the industrialized nations, current fiscal challenges may make it impossible to

continue unrestricted delivery of this treatment.

Another problem that was predicted at the time of development of ERT was

immunological response to recombinant human proteins. This was initially

identified in animals and subsequently recognized in patients receiving the ERT.

1.5.3. Human immune responses to recombinant proteins

A number of recombinant human proteins are in clinical use. A detailed review

on these products revealed that even though the immune response could

potentially reduce the efficacy of the products, in the majority of cases it was not

clinically significant (Porter 2001). Some of the factors which influence the

immunogenicity of recombinant proteins include denaturing of the molecules

during production, impurities, aggregation of particles, dose of administration,

route of administration, frequency of treatment and structural homology to

microorganism and non-human antigens (Richards ; Porter 2001). In the

majority of cases, IgG and IgM antibodies are produced in response to immune

stimulation and only occasionally IgE antibodies are implicated. Whilst IgG and

IgM antibodies are known to neutralize the effects of the humanized proteins,

IgE antibodies are mostly associated with allergic reactions including

anaphylaxis. One example of a humanized protein which is associated with

clinically significant immune response is that of recombinant human coagulation

factors used in Haemophilia patients. Antibodies against recombinant factor VIII

and factor IX (Haemophilia inhibitors) pose serious clinical problems which are

49

associated with significant morbidity and mortality. In a 3 year longitudinal study

on Haemophilia A patients, about a quarter of the treated patients developed

neutralizing antibodies. The risk of development of inhibitors in this cohort was

estimated to be 12%, 27% and 49 % at days 8, 10 and 25 of protein exposure

respectively (Bray, Gomperts et al. 1994). Antibodies from both IgG and IgM

class are implicated in inhibition of coagulation factors in vivo and in vitro

(Yoshioka, Fukutake et al. 2003). The functional assays performed on patients

with high titer antibodies demonstrate a considerable inhibition of replacement

therapy reducing the factor activity to less than 1% in some cases. In

Haemophilia patients with refractory immune responses, this poses a major

challenge in clinical management. Insulin is another example of replacement

therapy where immune responses can cause significant clinical impact. Immune

responses to both human and animal insulin can reduce the clinical efficacy of

the product. However, recombinant human insulin seems to generate milder

immune responses with less inhibition of administered insulin compared to the

porcine insulin (Spijker, Poortman et al. 1982; Fineberg, Galloway et al. 1983;

Fineberg, Galloway et al. 1983; Di Mario, Arduini et al. 1986). Studies on

various recombinant human proteins e.g. growth hormone (Gunnarsson and

Wilton 1987; Buzi, Buchanan et al. 1989; Takano, Shizume et al. 1989; Zeisel,

Lutz et al. 1992), granulocyte colony stimulating factor (G-CSF) (Laricchia-

Robbio, Moscato et al. 1997), erythropoietin (Nielsen and Thaysen 1989; Urra,

de la Torre et al. 1997) and interferon (Spiegel, Spicehandler et al. 1986) show

similar results. The immunogenicity of all these products is variable, influenced

by multiple factors (mentioned earlier) and sometimes results in reduced clinical

efficacy of the therapy delivered. The adverse clinical effects including treatment

failure are mediated by the ability of these antibodies to neutralize the delivered

protein and hence functional assay is a better predictor of the clinical outcome.

In haemophilia A and B the risk factors for development of neutralizing

antibodies (inhibitors) have been studied in great detail. It is estimated that

complete or near complete gene deletions result in inhibitor development in 20-

50

80% of the patients who are exposed to recombinant human factor VIII. Their

risk is significantly higher than those with smaller gene deletions resulting in

inhibitor development in less than 5% (Oldenburg and Pavlova 2006). This

difference in incidence is due to the presence of a small amount of residual

factor VIII produced by the patients who have minor gene deletions. This

constant exposure of the immune system to extremely low levels of

functional/non-functional factor VIII is sufficient to induce immune tolerance.

This residual protein is termed as cross reactive immunological material (CRIM).

However exceptions to this rule exist e.g. severe Haemophilia A patients with

intron 22 inversion only show modest inhibitor antibody response in the absence

of CRIM.

1.5.4. Immune response to ERT

As a part of pharmacovigilance, the pharmaceutical companies developing ERT

are required to monitor the immune response to their product. The generation of

immune antibodies was reported in phase I and II studies during development of

ERT.

In a randomized double blind placebo controlled study undertaken by Genzyme,

all patients exposed to Aldurazyme developed IgG immune response. The

median time to seroconversion was 25.8 days. The antibodies reported were

mostly of IgG class. Rarely, IgM and IgE antibodies were also reported. IgE

antibodies were mostly associated with severe systemic allergic reaction

(Lipinski, Lipinski et al. 2009). The patients with mild immune response showed

a robust decrease in urinary GAG whereas more severe immune responses

were associated with variable reduction in disease biomarker (Wraith, Clarke et

al. 2004). In a double blind placebo controlled study to evaluate the efficacy of

ERT in MPSI, 91% patients developed IgG response to laronidase during

treatment. The mean time to seroconversion was 52 days (range, 20 to 106).

Close to the end of the follow up period the majority of patients were

spontaneously tolerized. There were no significant differences between tolerized

51

and non-tolerized groups in terms of substrate reduction, clinical severity,

radiological and cardiac imaging. The authors suggested that seroconversion

did not significantly alter the tolerability and clinical efficacy of laronidase(Wraith,

Clarke et al. 2004). In another longitudinal study of 10 MPSI patients receiving

ERT (recombinant human -L-iduronidase) the antibody titers were studied to

evaluate the immune response to therapy and compared with normal controls

receiving the same treatment. At the start of treatment there was no difference

in antibody titers between the patient and the control group. Five of the patients

subsequently developed high titre immune response. By week 104, all patients

had low antibody titer or no antibodies. In this study there was no significant

difference in clinical outcome in the two treated groups regardless of their

immune response. The authors argued against the use of immune tolerance

induction in these patients due to potential morbidity associated with

conventional immune tolerance induction treatments (Kakavanos, Turner et al.

2003).

Various studies describing the effects of ERT in other LSDs e.g. MPSII (Hunters

Syndrome), MPSVI, Gaucher, Pompe and Fabry disease also showed

development of immune response in ERT treated patients occasionally

associated with adverse reactions necessitating cessation of treatment

(Richards, Olson et al. 1993; Rosenberg, Kingma et al. 1999; Amalfitano,

Bengur et al. 2001; Hunley, Corzo et al. 2004; Muenzer, Wraith et al. 2006;

Muenzer, Gucsavas-Calikoglu et al. 2007; Vedder, Linthorst et al. 2007; White,

Argento Martell et al. 2008; Tesmoingt, Lidove et al. 2009).

52

1.5.5. Functional and clinical impact of immune response

The antibody bound to the ERT can reduce its efficacy by various other

mechanisms including altered enzyme distribution, altered intracellular enzyme

trafficking and increased enzyme turnover (Brooks 1999). Whilst it is possible

that the in vitro enzyme inhibition does not reflect in vivo ERT failure subsequent

studies showed a correlation between the high antibody titre, in vitro enzyme

inhibition (catalytic activity and cellular uptake), reduced metabolic clearance of

GAG and adverse clinical outcome in some ERT treated LSDs. In a recent

publication Cox-Brinkman et al. described the effect of ERT and HSCT on

dermal fibroblast morphology in 12 patients with MPSI. The skin biopsies were

taken on regular intervals before and during 2 years of ERT as well as before

and 6 months after HSCT. This study showed that both ERT and HSCT

improved the tissue morphology of skin fibroblasts based on vacuolation score

of histological sections. However, three of the 12 patients in this study did not

show any improvement in their dermal fibroblast morphology. All of them had

IgG antibodies. Due to the small size of the study it was not possible to show

statistical correlation between treatment failure and incidence of these

antibodies. The follow up of the patients who received HSCT showed rapidly

declining levels of antibodies in these patients. These data are interesting as

they provide evidence of immune tolerance induction by HSCT and show that

the donor lymphocytes despite being educated in recipient reticuloendothelial

system, retain the donor immune response (Cox-Brinkman, van den Bergh

Weerman et al. 2010). In a recent study urinary GAG and HCII-T levels in 9

MPS I and 11 MPS II were evaluated and correlated with antibody titres. There

was a clear correlation between the antibody levels and HCII-T. These levels

improved at the onset of treatment but deteriorated after generation of immune

response often to values above the baseline. In contrast urinary GAG levels

were less responsive to antibody titres. In severe MPS I patients serum HCII-T

levels never corrected to normal in all patients treated with ERT (Clarke,

Hemmelgarn et al. 2012).

53

1.5.6. Effect of severity of immune response

Dickson et al. evaluated the effects of anti IDUA antibodies in animal models of

MPSI (dogs) treated with ERT and demonstrated that the presence of high titres

of anti-IDUA antibodies in serum reduced the uptake of ERT to less than 10%

compared to the serum from canines with low or negative titres. There was a

correlation between the high titre antibodies and urinary GAG excretion in this

study. Dose escalation and immune tolerance both reduced urinary GAG

excretion. Tissue histology revealed a remarkable improvement in the

appearance of kidneys and synovia but less response was observed in lymph

nodes and heart valves. Improvement in renal histology correlated with urinary

GAG excretion. The animals which had no antibodies or very low levels had

greater substrate reduction compared to those with high titre immune response.

Published data from other animal studies supports the findings in the above

study and shows that even though high titre immune responses reduce the

clinical efficacy of ERT, some clinical benefit can still be achieved when

compared to untreated animals (Dickson, Peinovich et al. 2008; Ponder 2008).

The response to ERT in the presence of antibodies is even more disappointing

in other tissues e.g. cartilage and heart valves (Shull, Kakkis et al. 1994). In a

study involving MPSI patients (70% of patients with iduronidase-null mutations),

all patients developed anti Iduronidase antibodies. Antibody titres greater than

1:10,000 were associated with a lower reduction in urinary GAG levels when

compared to the patients who had low or undetectable titres (Wraith, Beck et al.

2007).

CRIM negative infantile Pompe disease is another example of LSD where the

immune response to ERT appears to make the treatment ineffective. In a

longitudinal study the outcome of ERT (recombinant human acid alpha

glucosidase) was evaluated in 21 CRIM positive and 11 CRIM negative infantile

Pompe patients. Clinical evaluation of these patients included monitoring motor

development and assessment of cardiac function. Metabolic outcome and

54

immune response was monitored by substrate clearance and IgG antibody

ELISA assays. At 52 week follow up, nearly half of the CRIM negative and 5%

of CRIM positive children had died. After 27 months although none of the CRIM

negative patients survived, 80% of CRIM positive patients survived. In

comparison to the CRIM positive patient, those who were CRIM negative

generated the immune response quicker with higher titres which stayed at

higher levels during the follow up period. This study gave direct evidence of the

poor outcome of the disease in presence of high titre antibodies to ERT and

propensity of CRIM negative patients to develop a more severe and sustained

immune response. These data were in contrast to the initial data from MPSI

patients who were reported to develop immune tolerance spontaneously with

continuing infusions of ERT (Wang, Lozier et al. 2008; Kishnani, Goldenberg et

al. 2010). More recently, a study evaluating the impact of allo-immune response

in Pompe disease revealed a worse clinical response in CRIM positive patients

with high titer antibodies when compared to CRIM positive patients with low titer

immune response (Banugaria, Prater et al. 2011). In this study the overall

survival of CRIM negative patients was similar to CRIM positive patients with

high titer antibodies which underlines the significance of high titer immune

response. In a recent publication De Vries et al. described the immune response

and the effects of antibodies in a patient of Pompe disease. This 39 year old

patient experienced adverse reaction associated with infusion of ERT. Further

studies on antibody revealed significant inhibition of cellular uptake (on average

25% lower in the presence of patient serum compared to normal serum). These

antibodies failed to show quantifiable catalytic inhibition of enzyme in vitro but

there was some indirect evidence of enzyme activity inhibition by western blot

analysis and measurement of enzyme activity. This patient was reported to have

a worse clinical outcome compared to the reference subjects who did not have

significant immune response. The clinical assessment was done by monitoring

forced vital capacity, 6 minute walk, manual muscle testing and other isometric

muscle contractions. Based on the antigen antibody binding data, the authors

suggested that approximately 37.5% of the infused enzyme at recommended

55

dosage was captured by the antibodies at the time of maximum immune

response in this particular patient. (de Vries, van der Beek et al.).

Neutralizing antibodies have also been reported in other LSDs including MPS II,

Gaucher and Fabry disease which were associated with deterioration in clinical

response in some cases (Ponce, Moskovitz et al. 1997; Rubio, Kim et al. 2003;

Zhao, Bailey et al. 2003; Linthorst, Hollak et al. 2004; White, Argento Martell et

al. 2008).

1.5.7. Assays for detection and quantification of immune response

In order to understand the antibody response, its functional impact and

limitations in the existing practice of antibody testing, it is important to

comprehend the basic principles of antibody detection tests and functional

assays. A simple description of the principles of these assays is as follows.

1) Assays for detection of the immune response

The recommendations for the development of immunological assays to detect

antibodies against any therapeutic protein also apply to ERT. These have been

published previously (Mire-Sluis, Barrett et al. 2004; Shankar, Shores et al.

2006). The assays should include the use of a highly sensitive technique e.g.

ELISA. A positive result then needs to be verified to ensure that the positive

response is due to the antibodies and is specific to the antigen (ERT in this

case). This can be achieved by performing specific assays e.g.

immunoprecipitation assays or Western blot analysis. Briefly, during ELISA the

antigen (recombinant enzyme) is fixed on a solid support i.e. ELISA plate either

specifically via capture by a specific antibody in a sandwich ELISA, or non-

specifically by direct binding of the antigen to the surface. Patient serum is then

added to the fixed antigen to capture antibodies. The antibodies in human

serum bind to the antigen. A detection antibody which is usually an antihuman

antibody is exposed to the plates which form a covalent bond to the antigen

56

antibody complex. After exposure to a specific substrate the complex produces

a visible signal which is read by light absorption or transmission. IgG, IgM and

IgE antihuman antibodies can be used to detect serum antibodies of respective

classes. This assay needs optimisation and validation which involves

confirmation of specificity of the ELISA assay by immunoprecipitation tests or

Western blot analysis. The presence of antibody in patient serum is not an

evidence of enzyme inhibition. This requires functional enzyme assays.

2) Catalytic enzyme inhibition assay

This assay determines neutralization of catalytic activity of enzyme by the

antibodies in the patient serum. This can be performed by various different

techniques and there is no consensus on the most appropriate method. The

demonstration of enzyme activity inhibition would inevitably require mixing

studies. This can be achieved by mixing a patient’s serum or antibodies isolated

from serum with a validated potency enzyme assay. The inhibition of enzyme

activity of the solution after mixing the patient’s serum gives a quantifiable

measure of direct catalytic enzyme inhibition. A standard should be established

and validated for the comparison and quantification of enzyme activity inhibition.

Enzymes from various sources (recombinant human and innate) can be used

for inhibition studies.

3) Cellular uptake inhibition assay

This assay differs from enzyme activity inhibition as it evaluates a different effect

of antibody i.e. its ability to inhibit the uptake of enzyme by enzyme deficient

cells. This is determined by developing a standard cell line for each disease

group (usually non FcR expressing fibroblast). These cells are then incubated

with enzyme solution in the presence and absence of patient serum (with

antibodies). The uptake of enzyme by the cells can be measured by various

methods. Broadly, it can be measured either directly by flow cytometric analysis

of the cells to detect the labeled enzyme present in the fibroblasts, or indirectly

by measuring the enzyme activity of cell lysate. The comparison of enzyme

57

uptake by the cells in the presence or absence of antibodies gives a measure of

cellular uptake inhibition.

1.5.8. Limitations of existing assays

Currently the assays for antibody detection, quantification and assessment of

functional impact are being carried out by the pharmaceutical companies. There

is no consensus on methodology, standardisation or reporting of these assays.

A number of different ELISA techniques have been used for detection of

antibodies in patient serum which are not comparable and lack a robust external

quality assessment mechanism. Recently, efforts have been made to optimize

and standardize these assays (Sellos-Moura, Barzegar et al. 2011). It is vital

that the catalytic inhibition assays actually measure the drop in catalytic activity

of the enzyme after the mixing of enzyme solution with the antibodies from

patient serum. In some instances binding and precipitation of the enzyme by the

antibodies has been implied as catalytic inhibition. Whilst antigen antibody

binding will inevitably be required to inhibit the catalytic activity, it may not do so

in all cases due to the polyclonal nature of IgG antibodies. The substrates used

to detect catalytic inhibition are artificial and might not reflect true in vivo

enzyme inhibition. Similarly there is no standard cell line in use, to study the

cellular uptake inhibition for various diseases and hence it is difficult to compare

the results from various centres. This problem is compounded by a lack of

efficient and reliable biomarkers which predict the treatment failure

instantaneously.

1.6. Diagnosis, monitoring and disease biomarkers

LSDs are diagnosed by direct enzyme assays. This is available for most of the

disorders but in a small minority the diagnosis is made on clinical findings

coupled with radiological investigations, electroretinogram, tissue biopsy and

mutation analysis. Enzyme levels can be tested in peripheral blood (serum,

leucocytes), tissue fibroblasts and urine (Meikle, Hopwood et al. 1999; Wilcox

58

2004). In most disorders it is possible to make a prenatal diagnosis by chorionic

villus sampling or amniocentesis. Carriers should only be identified by mutation

analysis as they might have normal or near normal levels of enzyme which

sometimes are difficult to differentiate from normal individuals.

Urinary GAG assay has been in use for some time as a biomarker of the

disease. In the clinical settings enzyme levels and urinary GAG are used to

confirm the diagnosis in new patients. More recently urinary dermatan sulphate

(DS) to chondroitin sulphate (CS) ratio was found to be a superior biomarker in

MPSI compared to urinary GAG (Church, Tylee et al. 2007). The development

of heparin cofactor II- thrombin (HCII-T) as a reliable biomarker in MPSI, II, III,

VI and VII (serum and dried blood spot) has opened new avenues for using this

biomarker as a cheap neonatal screening tool and a biomarker to study the

disease progression in these illnesses (Langford-Smith, Arasaradnam et al.

2010; Langford-Smith, Mercer et al. 2010).

1.7. Immune tolerance induction

Recently, immunogenicity was recognized as one of the most important

“modifiable factors” compromising the long term efficacy of ERT (Lacana, Yao et

al. 2012). This has resulted in increased emphasis now placed on development

of strategies to induce immune tolerance in recipients of ERT. Development of

immune tolerance is a complex process. Most of the immune responses

generated by adaptive immune system rely on B and T lymphocytes. After

sensitization and stimulation by helper T cells, B cells evolve to form antibody

secreting cells (plasma cells). However, antibodies generated by the human

immune system in response to some infused protein are independent of T cell

interaction. B1B cells, which are not regulated by helper T cells are capable of

producing antibodies without interaction with helper T cells. As most of the IgG

antibodies produced in response to recombinant human protein belong to IgG1

and IgG4 classes, it is widely accepted that the helper T cell dependent

mechanisms are also involved in generating antibody responses (Herzog, Fields

et al. 2002; Withers, Fiorini et al. 2007). This makes both B and T lymphocytes a

59

target for immune tolerance induction in these patients. Various immune

tolerance induction regimens have been tried to improve the metabolic outcome

of ERT. Conventional tolerance regimens used in severe Haemophilia patients

with inhibitors do not show promising results in ERT treated LSD patients with

neutralizing antibodies. Serious side effects e.g. systemic allergic reactions and

nephrotic syndrome have been reported in patients who were treated with

escalating doses of ERT to induce immune tolerance (Hunley, Corzo et al.

2004).

Immune tolerance induction regimens used in clinical practice can be

categorized into two groups. The first involves immune suppression or

myelosuppression which impedes the adaptive immune response by either

cellular inhibition or switching off the humoral component of the immune system.

The second includes repeated exposure of the immune system to the deficient

protein to achieve tolerance. The potential mechanism by which the immune

system becomes tolerized using the former technique is not clear but it probably

involves steric hindrance, inhibition of degradation of infused protein or

immunomodulation (Astermark 2011). In LSDs, this hypo-sensitization can

either be achieved by regular infusion of the exogenous recombinant enzyme or

delivery of innate enzyme in the form of cellular therapy (HSCT) which delivers

enzyme produced by the donor cells after engraftment. The two strategies can

be used concurrently to enhance and expedite immune tolerance induction. In

the past, gene therapy was incorporated into this strategy to achieve immune

tolerance induction. In animal models of Haemophilia successful immune

tolerance using HSCT based gene transfer has been described (Bigger, Siapati

et al. 2006; Pichard, Bellodi-Privato et al. 2006). More recently HSCT based

gene therapy was evaluated in the murine model of Pompe disease. Lentivirus

mediated gene transfer was able to partially correct the phenotype of the

disease. Expression of deficient protein in haemopoietic precursor cells did not

produce immune response in immunocompetent mice but instead tolerized them

60

to the delivered ERT (Douillard-Guilloux, Richard et al. 2009). A similar strategy

has also been employed in MPSIIIA (Langford-Smith, Wilkinson et al. 2012).

Whilst most LSDs are slowly progressive disorders where a gradual induction

regimen is a suitable management strategy, in other disorders e.g. infantile

Pompe this is not acceptable. In infantile Pompe the development of high titre

antibody to ERT is invariably fatal and the development of a reliable, successful

and quick immune tolerance induction regimen is crucial. In order to achieve

this, a number of conventional immune tolerance induction treatments were

studied in a knock-out mouse model of Pompe disease. In this study

mycophenolate mofetil, methotrexate and cyclosporine with azathioprine were

evaluated as immune tolerance induction regimens. Empirical treatment with

low dose methotrexate (3 weekly) started within 24 hours of administration of

the first dose of recombinant human acid alpha glucosidase was the only

successful regimens. This treatment prevented the development of high titre

antibody response to the ERT. The exact mechanism of action of methotrexate

in this context is not clear. Further experiments on these mice revealed B1 B

cell dysregulation as demonstrated by the consistent expansion of paritoneal

B1B cell clones. However it is not yet clear how this cellular expansion leads to

the development of immune tolerance induction to ERT (Joseph, Munroe et al.

2008). In another study on a canine model of MPSI a different induction therapy

was found to be beneficial. In this study a regimen consisting of cyclosporine A

and azathioprine combined with low dose weekly infusions of recombinant

human alpha-L-iduronidase (strategy 1 and 2 combined) was evaluated. The

induction regimen was given over a period of 60 days which successfully

prevented a high titre immune response to therapeutic doses of enzyme (once

weekly infusion) for a period of 6 months. During this study ciclosporine levels

were also monitored and sub-therapeutic cyclosporine was shown to have an

inferior outcome (Kakkis, Lester et al. 2004). In the past, immune tolerance was

induced in a patient with Gaucher disease by using a combination of plasma

61

exchange, cyclophosphamide, intravenous immunoglobulins and large doses of

Ceredase (Brady, Murray et al. 1997).

Whilst more complex regimens and combinations of immune suppression might

be promising, they increase the risks associated with therapy. In Haemophilia,

immune tolerance induction success rates vary between 40-80 per cent

depending upon the therapeutic regimens. These regimens include low dose

chemotherapy, administration of high doses of FVIII twice daily (Bonn protocol),

low FVIII doses three times weekly (van Creveld protocol), immune adsorption

followed by immune suppression (Malmö protocol) and modified Malmo-Bonn

regimen (acquired Haemophilia). However the relapse rates are high. More

complex regimens using combination treatments and immune suppression are

associated with significant morbidity. Most of the side effects are due to

immunosuppression but more serious adverse reactions including anaphylaxis

have been reported during the use of escalating doses of replacement therapy

in an effort to achieve immune tolerance (Berntorp, Astermark et al. 2000;

Carlborg, Astermark et al. 2000; Kreuz, Ettingshausen et al. 2003; Wight,

Paisley et al. 2003; Curry, Misbah et al. 2007). In a study on a knock-out mouse

model of Pompe disease, various immune tolerance regimens including

mycophenolate mofetil, methotrexate and cyclosporine with azathioprine were

evaluated. Empirical treatment with low dose methotrexate successfully induced

immune tolerance (Joseph, Munroe et al. 2008). In another study on a canine

model of MPS I, cyclosporine A (within therapeutic levels) and azathioprine

combined with low dose weekly infusions of laronidase (strategy 1 and 2

combined) successfully prevented development of high titer antibody response

(Kakkis, Lester et al. 2004). A successful regimen involving combination of

plasma exchange, cyclophosphamide, intravenous immunoglobulins and large

doses of Ceredase (Brady, Murray et al. 1997) is described in Gaucher disease

patients.

Rapid and effective immune tolerance induction is critical in some infantile LSDs

where development of high titer antibodies leads to catastrophic outcome very

rapidly. Intensification of the immune tolerance induction regimens by using

62

combination chemotherapies could achieve rapid immune tolerance, but there is

a risk of increased morbidity associated with these approaches. Use of novel

treatment approaches e.g. monoclonal antibodies (anti-CD20 antibody) have

been successfully used to induce immune tolerance in some congenital

disorders (Franchini, Mengoli et al. 2008; Barnes, Davis et al. 2010) but there is

very little data on the use of these treatment options in ERT treated LSD

patients (Mendelsohn, Messinger et al. 2009). Recently Messinger et al reported

successful use of a combination of Rituximab (anti-CD 20 antibody),

methotrexate and immunoglobulins to induce immune tolerance in two CRIM

negative Pompe disease patients. In addition, two patients (both CRIM

negative) were given prophylactic Rituximab and Methotrexate and remain

tolerant to recombinant human alpha glucosidase at 18 and 35 months of age

(Messinger, Mendelsohn et al. 2012). Addition of bortezomib to conventional

immunomodulatory immune tolerance strategy showed promising results in

Pompe disease patients (Banugaria, Prater et al. 2012).

HSCT has previously been used to eradicate the immune response in a number

of auto-immune disorders such as systemic lupus erythematosus (Jayne,

Passweg et al. 2004; Lisukov, Sizikova et al. 2004; Burt, Traynor et al. 2006)

and rheumatoid arthritis (Snowden, Passweg et al. 2004; Teng, Verburg et al.

2005). In 2010, Uprichard et.al. published a case report of a 22 year old patient

with severe Haemophilia A with refractory high titre inhibitor to factor VIII

replacement therapy, who received allogeneic HSCT to induce immune

tolerance (Uprichard, Dazzi et al. 2010). Unfortunately, this patient died of

Klebsiella pneumonia septicaemia on day 46 post transplantation. However,

during the very brief follow up period, the inhibitor to infused factor VIII re-

emerged; suggesting a persistence of immune response mediated by memory T

cells which were not completely ablated by reduced intensity conditioning. The

authors suggested that a subsequent donor lymphocyte infusion could eradicate

this immune response completely as is shown in animal models (Van

Wijmeersch, Sprangers et al. 2007). Currently there is no such data available on

the role of allogeneic HSCT in LSDs. Allogeneic HSCT could induce immune

63

tolerance in LSD patients with refractory immune response and bring both

replacement of the defective enzyme (through hematopoietic stem cells and

their differentiated progeny) and eradication of inhibitory immune response. With

the elimination of the recipient immune system as part of conditioning for HSCT,

enzyme-reactive B and T cells are removed. Following the acute period of

immune reconstitution after the transplant, in which the risk of GvHD is

managed by immune suppression, newly developing donor T and B cells would

display tolerance to specific donor and host proteins.

Unlike many other allo and auto immune disorders where novel treatment

approaches e.g. monoclonal antibodies (Rituximab, Campath) have been tested

in large patient series, there is very little data on the use of these novel

treatment options in ERT treated LSD patients.

1.8. Project aims

1.8.1. Development of quantitative and functional immune assays for

LSDs

The first aim of this research project was to develop quantitative and functional

immune assays in MPSI, MPSVI and Pompe disease patients who were

exposed to ERT. In view of high sensitivity and specificity, ELISA was selected

as the basis for the detection assay. Two different types of functional assays

were to be developed in order to evaluate inhibition of catalytic enzyme activity

and cellular uptake inhibition.

64

1.8.2. To study the incidence and impact of immune response in MPSIH

patients treated with ERT and HSCT

The current practice of delivering ERT to MPS I patients around the time of

allogeneic HSCT, afforded us the opportunity to study the kinetics of the allo-

immune response in Aldurazyme (recombinant human IDUA) treated patients

with MPS I. The aim was to study the incidence of allo-immune response with

IgG ELISA and assess the functional impact of allo-antibodies in recipients of

ERT and HSCT. We also wanted to explore HSCT as an immune tolerance

induction strategy.

1.8.3. To evaluate the nature and incidence of immune response in other

LSDs

Based on immune assays developed in aim 1, all patient samples collected over

the period of research and archive samples, collected prior to the start of study,

were to be evaluated to explore the incidence of functional nature of immune

response in MPSI HS, MPS VI and Pompe disease patients. This would include

patients on long term ERT.

65

2. Materials and methods

66

2.1. Patients and sample preparation

Blood samples from LSD patients who were treated and followed up in Royal

Manchester Children’s Hospital over a period of four years were collected with

informed consent under ethical permission REC 08H101063. Patient blood

samples were processed within 6 hours of collection. The serum samples (with

no anti-coagulant) were centrifuged at 3000 G at room temperature and then

serum was separated and stored at -80oC. Samples were grouped into

longitudinal series (with two or more time points) and cross sectional series

(single time point) for each LSD.

2.2. Principles of Immune assays

2.2.1. ELISA

During this assay, an antigen (enzyme) is fixed on a surface (EIA plate)

overnight. The plate is then washed with washing buffer and blocked by a

blocking solution containing human serum albumin. This ensures that the non-

specific binding sites, on the surface, are blocked and reduces the background

interference. After thoroughly washing the plates, a primary antibody (patient

serum) is applied to the antigen. The plates are washed between every step to

minimize contamination. A secondary antibody (anti-human IgG antibody) is

then applied to the EIA plates. These antibodies are conjugated with a

substance which changes colour on exposure to a specific substrate. This

reaction can be quantified accurately by measurement of light absorption as

shown in the figure 2.1. Due to the unavailability of commercially available

standardized human IgG antibodies to respective enzymes, a standard curve

could not be created in the assays. Therefore, a negative control was defined

67

for each dilution. For each disorder, a different set of experiments were

undertaken to define a negative cut off and optimize enzyme concentrations

used for the ELISA. This is elaborated in respective sections in chapter 3.

Figure 2.1 IgG Enzyme linked immunosorbent assay

Antigen (enzyme) is applied to a surface (EIA plate). Primary antibodies (patient serum) bind to the antigen. These antibodies are detected by a secondary antibody (HRP conjugated, anti-human IgG antibodies) which changes the colour of the reaction when exposed to the substrate (OPD).

2.2.2. Catalytic inhibition

For all disorders studied in this project, functional enzyme assays were

optimized in 96 well plates. These assays were then modified to develop mixing

assays. The inhibitory antibodies in patient serum may block the catalytic site of

the enzyme. Mixing of patient serum with an enzyme solution of known enzyme

68

concentration, will then inhibit the enzyme activity of the solution. This inhibition

can be quantified by comparing enzyme activity of the solution in the absence of

serum and presence of normal serum and patient serum as shown in figure 2.2.

An extensive series of experiments had to be undertaken, in order to define

optimum conditions for each disorder.

Figure 2.2 Catalytic inhibition assay

Catalytic inhibition assay. Antibodies in patient serum bind to the enzyme and reduce the ability of enzyme to breakdown artificial substr ate resulting in reduced release of 4MU. This is then compared to a standard (normal human serum) to quantify the inhibition of enzyme activity.

69

2.2.3. Cellular uptake inhibition

Cellular uptake inhibition assays are based on functional enzyme activity assay

and hence they determine the ability of the antibodies to block the cellular

uptake of functionally active enzyme. For each disorder, human fibroblasts from

patients with severe disease phenotype (deficient in enzyme) were acquired and

maintained in culture media. These cells were seeded into 6 well culture plates.

Under optimum conditions, these fibroblasts were exposed to the deficient

enzyme in the presence and absence of antibodies (in patient sera). The cells

were subsequently lysed by freezing and sonication. The amount of enzyme

within the cells was accurately measured by enzyme assays. Cellular uptake

inhibition was quantified by comparing the amount of enzyme delivered into the

cell in the presence and absence of antibodies (see figure 2.3). For various

disorders, the experiment conditions such as exposure time and enzyme

concentrations had to be changed considerably to elicit cellular uptake inhibition

as described in detail in chapter 3.

70

Figure 2.3 Cellular uptake inhibition assay

Cellular uptake inhibition assay. Antibodies in patient serum bind to the enzyme and reduce its uptake by enzyme deficient patient fibroblasts. The cells are lysed and the enzyme activity is measured accurately. The comparison of enzyme activity in fibroblasts in the presence and absence of antibodies gives an accurate measure of inhibition of enzyme uptake by antibodies.

71

2.3. MPSI

2.3.1. IgG ELISA

In this assay, the antigen (recombinant human IDUA, Aldurazyme), is fixed on

the enzyme immunoassay (EIA) plate. Antibodies present in patient serum,

which are specific to the enzyme (Aldurazyme), bind to this antigen. These

antibodies are then detected by secondary anti-human IgG antibodies

conjugated with a substrate.

In our assay, 96 well EIA plates were coated with enzyme-carbonate solution

(10µg/ml in 0.1M NaHCO3, pH8.5) overnight and blocked with blocking agent

containing 1% HSA. Patient serum (50μl) was added (two fold serial dilutions,

see figure 2.4) to each well in duplicate and incubated at room temperature for 1

hour.

Figure 2.4 Preparation of serial dilution of specimens

One hundred microlitres of IgG goat anti human Horseradish peroxidase

antibody (1:5000 dilutions) was added to each well and incubated at room

temperature (RT) for 1 hour. Following this period, 100µl o-Phenylenediamine

dihydrochloride (OPD) solution was added to each well and left for 10 minutes in

a dark place. The reaction was stopped by 2.5M H2SO4 (50µl into each well) and

the light absorbance was read at 492nm immediately. The normal range was

determined for each dilution by testing 13 normal sera (Normal volunteers). A

positive cut off value was defined as two SD above the absorbance value for the

normal sera at that concentration. To quantify ELISA, the lowest titre with

72

absorbance value above the cut off was reported as positive serum dilution for

that patient sample. Positive responses were confirmed by western blot to show

the specificity of the IgG antibody to Aldurazyme.

2.3.2. Enzyme activity assay

Our catalytic inhibition assays were based on functional enzyme activity assay

measured by using 4-Methylumbelliferyl (4MU) α-L-iduronide substrate

(Glycosynth, Warrington, UK) as previously described (Stirling, Robinson et al.

1978; Kakkis, Matynia et al. 1994). The α-L-iduronidase cleaves α-L-iduronic

acid from the substrate to yield the fluorescent product 4-methylumbelliferone

(4-MU). The liberated 4-methylumbelliferone is measured using a luminescence

spectrophotometer against a standard of known concentration.

4-methylumbelliferyl-α-L-iduronide 4-methylumbelliferone +

iduronic acid

The assay was performed in duplicate. Samples were prepared in a 96 well

plate. Patient sample (20 µl) and 20µl artificial substrate (4-methylumbel1iferyl-

α-L-iduronide; Glycosynth) in substrate buffer (0.4M formate buffer, pH 3.5,

0.9%NaCl) were added into each well. The plate was wrapped in cling film and

covered with aluminum foil to prevent light interference. The plate was

incubated in a shaking incubator at 37°C for 1 hour and reaction was stopped by

adding 160 µl of stop buffer (0.2M sodium carbonate, 0.1M sodium hydrogen

carbonate). The fluorescence generated was read at excitation 360nm

(wavelength absorbed by the substrate) and emission of 450nm (wavelength

emitted). This fluorescence was then compared to a standard by developing a

standard curve using a fluorescent standard as shown in table 2.1.

α-L-iduronidase

73

Concentration

4-MU

µl Stock 4-MU

(66.67 µM)

µl of stop

buffer

30 µM 90 µl 110 µl

20 µM 60 µl 140 µl

10 µM 30 µl 170 µl

5 µM 15 µl 185 µl

2 µM 6 µl 194 µl

1 µM 3 µl 197 µl

0.5 µM 1.5 µl 198.5 µl

0 µM 0 µl 200 µl

Table 2.1 Fluorescent standard

2.3.3. Mixing assay (quantification of catalytic inhibition)

The above protocol was modified to develop and optimize mixing assays which

determine inhibition of enzyme activity by patient sera. This assay determines

the ability of a patient serum (with anti IDUA IgG antibodies) to inhibit the

enzyme activity of Aldurazyme solution with a known enzyme concentration.

This is then compared to the normal serum and baseline enzyme activity (no

serum) to quantify the percentage enzyme inhibition. The enzyme activity is

measured by functional enzyme activity assays using the artificial substrate 4-

methylumbelliferyl-α-L-iduronide as previously described (see figure 2.2).

The assay was performed in duplicate on the highest titre patient sample in the

longitudinal series. A series of two fold dilutions (starting from 30ng/ml to

optimize the catalytic inhibition) of recombinant human α-L-iduronidase

(Aldurazyme, Genzyme, MA) in dilution buffer (PBS, 0.05% Tween, 0.01% HSA)

were used to assess inhibition of enzyme activity. Ten microlitres of each

enzyme solution was then mixed with the same volume of patient sera and

normal sera in 96 well black plates to minimize the auto fluorescence and

background light scatter. The sera were added at fixed dilutions (1:4) and

74

incubated at room temperature in a shaking incubator for two hours. 4MU

substrate (20 µl) in substrate buffer (0.4M formate buffer, pH 3.5, 0.9%NaCl)

was added to each well and incubated for an hour at room temperature in the

dark. The enzyme activity in the absence and presence of sera (for patient

serum and normal serum) was measured. We observed some inhibition of

enzyme activity with normal serum and hence for each experiment normal

serum was used as a standard to compare and quantify the inhibition caused by

the antibodies in patient sera. The enzyme activity in the presence of patient

serum was also compared with enzyme activity of the plain enzyme solution for

each dilution. We also used IDUA from human embryonic kidney cells (HEK)

and mouse liver cells to assess the inhibition of innate enzyme by patient sera.

Enzyme activity in normal serum and patient serum was also measured. This

was then subtracted from the observed enzyme activity of the solution to

remove the background enzyme activity which ensured that the observed

activity in Aldurazyme solution was due to the recombinant enzyme only.

The % inhibition of the catalytic activity for each enzyme dilution was measured

as follows,

% Inhibition= 100-100xEAPs- BEAPs

EANs- BEANs

EAPs: Enzyme activity in the presence of patient serum

EANs: Enzyme activity in the presence of normal serum

BEAPs: Background enzyme activity in patient serum

BEANs: Background enzyme activity in normal serum

75


IgG antibodies in patient serum can bind to the infused enzyme at 6MP site and

inhibit the uptake of enzyme by the patient cells. This assay determines the

inhibition of enzyme uptake by non-FCR expressing, IDUA deficient fibroblasts

in the presence of antibodies. Enzyme activity in cells is measured by using 4-

Methylumbelliferyl α-L-iduronide as described earlier (see figure 2.3).

MPSI fibroblasts (MPSI-A171) from a Hurler patient were established and

maintained in DMEM, 10% FBS, 1% glutamine at 37oC and 5% CO2. The

assays were performed in duplicates. Six well culture plates were seeded with

1.5x105 fibroblasts into each of the six wells. The cellular uptake inhibition was

performed once the wells were 90-95% confluent, 24-48 hours later. The

enzyme was diluted in culture medium (DMEM with 1% glutamine) to below ½

Km for Aldurazyme (100 ng/ml). Patient serum or normal serum was added in a

volume of 10 µl (1 in 100 dilutions) to the diluted enzyme solution (1000 µl

volume) and incubated for two hours at room temperature. The culture medium

in seeded wells was replaced with the enzyme mixed medium incubated with

either normal or patient serum. The cells were incubated for another one hour in

5%CO2 at 37oC. The cells were then washed with PBS and harvested by

trypsinization. The cells were resuspended and washed twice with PBS and final

suspension made in homogenization buffer (0.5M NaCl/0.02M Tris pH7-7.5).

Following a cycle of freeze-thaw, the cells were sonicated, centrifuged at 2045G

for 10 minutes. The supernatant was tested for enzyme activity using 4MU

assay (described earlier). The protein concentration was determined by

Bicinchoninic acid (BCA) assay as described previously(Smith, Krohn et al.

1985). No uptake inhibition was observed with normal sera and hence the per

cent inhibition was calculated against plain enzyme solution as follows:

% Inhibition= 100-

100xEnzyme activity of lysate incubated in the presence of patient serum

Enzyme activity of lysate incubated with enzyme

76

2.4. Pompe disease

2.4.1. ELISA

96 well flat bottomed EIA plates were coated with 50µl/well enzyme carbonate

solution (1µg/ml myozyme in 0.1M NaHCO3, pH8.5) and stored at 4oC

overnight. Excess enzyme-carbonate solution was carefully aspirated next day

and the plate was washed gently 5x with wash buffer (PBS, 0.1% Tween,

pH7.4).To each well, 200ul of blocking Agent (1% w/v HSA in 0.02M Tris /HCl,

0.25M NaCl buffer pH7.0) was added and the plate stored at RT for 1 hr. After

carefully aspirating the excess blocking agent, plates were washed again with

wash buffer x 5. Using dilution buffer (PBS, 0.05% Tween, 0.01% HAS), 2 fold

dilutions (16 dilutions in total) of specimens to be tested, positive control and

negative control were prepared.

To each well, 50µl of standard, control or sample were added and plates

covered with an adhesive cover slip and incubated at RT for 1 hr. After

aspirating the tested samples the plates were washed 5x with wash buffer. IgG

goat anti human HRP antibody was added (100µl) at a 1:5000 dilution to each

well and incubated at RT for 1 hr. At the end of incubation, the plates were

washed 5x and 100µl of OPD was added to each well. The plates were kept for

10 minutes at RT in a dark place. The reaction was stopped by adding 50µl

2.5M H2SO4 and the absorbance of the plate was read at 492nm immediately.

2.4.2. Enzyme assay in 96 well plate

The activity of the lysosomal enzyme α-glucosidase was measured using an

artificial substrate 4-methylumbelliferyl-α-D-glucopyranoside. The assay was

based on the previously described method (Cooper, Hatton et al. 1988) which

was modified and optimized for a 96 well plate. Enzyme α-glucosidase cleaves

77

α-D-glucose from the substrate to yield the fluorescent product 4-

methylumbelliferone. The liberated 4-methylumbelliferone is then measured

using a luminescence spectrophotometer against a standard of known

concentration.

4-methylumbelliferyl-α-D-glucopyranoside 4-methylumbelliferone

+ glucose

The enzyme α-glucosidase is a glycosidase which hydrolyses terminal α1-4 and

α1-6 glucose residues in the degradation of the glycogen within lysosomes. The

assay was performed in duplicate. Samples were prepared in a 96 well plate.

Specimens to be tested and artificial substrate (4-methylumbelliferyl-α-D-

glucopyranoside), prepared in substrate buffer (0.1M citrate/0.2M phosphate

buffer at pH 4.0), were added to each well of the plate in equal volumes (20µl).

The plate was wrapped in cling film and covered with aluminum foil to prevent

light interference. The plate was incubated in a shaking incubator at 37°C for 30

minutes and reaction was stopped by adding 160 µl of stop buffer (0.2M sodium

carbonate, 0.1M sodium hydrogen carbonate). The fluorescence generated was

read at excitation 360nm and emission of 450nm. This fluorescence was then

compared to a standard by developing a standard curve using a fluorescent

standard as described previously

2.4.3. Catalytic inhibition assay

Enzyme assays were modified to develop mixing assays in order to determine

the inhibition of enzyme activity by patient serum. The assay was performed in

duplicate on the highest titre patient sample. A series of two fold dilutions

(starting from 12.5µg/ml) of recombinant human alglucosidase α (Myozyme) in

dilution buffer (PBS, 0.05% Tween, 0.01% HSA) were used to assess inhibition

α-glucosidase

78

of enzyme activity. Ten microlitres of each enzyme solution was then mixed with

the same volume of patient sera and normal sera in 96 well black plates. The

sera were added at fixed dilutions (1:4) and incubated at 4C for 24 hours. At the

end of first incubation, 4MU substrate (20 µl) in substrate buffer (0.4M formate

buffer, pH 3.5, 0.9%NaCl), was added to each well and incubated for 30

minutes at room temperature in the dark. The enzyme activity in the absence

and presence of sera (for patient serum and normal serum) was measured. The

enzyme activity in the presence of patient serum was also compared with

enzyme activity of the plain enzyme solution for each dilution. Background

enzyme activity was measured in normal and patient serum.


as follows,


EANs- BEANs






This assay determines the inhibition of enzyme uptake by, acid alpha

glucosidase deficient fibroblasts in the presence of antibodies. Fibroblasts from

a Pompe disease patient were established and maintained in DMEM, 10% FBS,

1% glutamine at 37oC and 5% CO2. The assays were performed in duplicates.

Six well culture plates were seeded with 1.5x105 fibroblasts into each of the six

wells. The cellular uptake inhibition was performed once the wells were 90-95%

confluent, 24-48 hours later. The enzyme (Myozyme) was diluted in culture

medium (DMEM with 1% glutamine) at concentrations of 12.5µg/ml and

6.25µg/ml to ensure penetration of enzyme into the enzyme deficient cells

(Yang, Kikuchi et al. 1998). Patient serum or normal serum was added in a

79

volume of 10 µl (1 in 100 dilutions) to the diluted enzyme solution (1000 µl

volume) and incubated for one hour at room temperature. The culture medium

in seeded wells was replaced with the enzyme mixed medium incubated with

either normal or patient serum. The cells were incubated for another six hours in

5%CO2 at 37oC. The cells were then washed with PBS and harvested by

trypsinization. The cells were resuspended and washed twice with PBS and final

suspension made in homogenization buffer (0.5M NaCl/0.02M Tris pH7-7.5).

Following a cycle of freeze-thaw, the cells were sonicated, centrifuged at 2045G

for 10 minutes. The supernatant was tested for enzyme activity using 4MU

assay (described earlier). The protein concentration was determined by

Bicinchoninic acid (BCA) assay (Smith, Krohn et al. 1985). The percent

inhibition was calculated against plain enzyme solution as follows:

% Inhibition= 100-

(100xEnzyme activity of lysate incubated in the presence of patient serum

Enzyme activity of lysate incubated with enzyme )

2.5. MPS VI

2.5.1. ELISA


solution (10µg/ml of Naglazyme in 0.1M NaHCO3, pH8.5) and stored at 4oC

overnight. After aspirating the excess enzyme next day the plate was washed

gently 5x with wash buffer (PBS, 0.1% Tween, pH7.4). 200ul of blocking Agent

(1% w/v HSA in 0.02M Tris /HCl, 0.25M NaCl buffer pH7.0) was added to each

well and plate stored at RT for 1 hr. The plates were then washed again with

wash buffer x 5. Using dilution buffer (PBS, 0.05% Tween, 0.01% HAS), two fold

dilutions (16 dilutions in total) of all specimens to be tested, were prepared. To

each well, 50µl of standard, control or sample were added and plates were

covered with an adhesive cover slip and incubated at RT for 1 hr. After

aspirating the tested samples the plates were washed 5x with wash buffer. IgG

goat anti human HRP antibody was added (100µl) at a 1:5000 dilution to each

80

well and incubated at RT for 1 hr. At the end of incubation, the plates were

washed 5x and 100µl of OPD was added to each well. The plates were kept for

10 minutes at RT in a dark place. The reaction was stopped by adding 50µl

2.5M H2SO4 and the absorbance of the plate was read at 492nm immediately.

2.5.2. Enzyme assay in 96 well plates

The activity of the lysosomal enzyme arylsulphatase B (N-acetylgalactosamine-

4-sulphatase) was measured using the artificial substrate 4-methylumbelliferyl-

sulphate. Previously described assays (Fluharty, Stevens et al. 1974) were

modified to measure enzyme activity using 96 well plates. The arylsulphatase B

cleaves sulphate from the substrate to yield the fluorescent product 4-

methylumbelliferone. The liberated product is then measured using a

luminescence spectrophotometer against a standard of known concentration.

Arylsulphatase B

4-methylumbelliferyl-sulphate 4-methylumbelliferone+

sulphate

This assay measures the collective activity of arylsulphatase B and its

isoenzymes including arylsulphatase A and arylsulphatase C, hence

measurement of Arylsulphatase B requires separation of arylsulphatase B by

chromatography. However, this step in not necessary during the mixing assays

where purified recombinant human Arylsuphatase B (Naglazyme) is used and

there is no contamination of other isoenzymes at any stage.

The enzyme activity assay was performed in duplicate. Specimens to be tested

and artificial substrate (4-methylumbelliferyl-sulphate), prepared in substrate

buffer (0.1M acetate buffer pH 5.4) at a concentration of 5mg/ml, were added to

each well of the plate in equal volumes (20µl). The plate was wrapped in cling

film and covered with aluminum foil to prevent light interference. The plate was

incubated in a shaking incubator at 37°C for 2 hours and reaction was stopped

81

by adding 160 µl of stop buffer (0.2M sodium carbonate, 0.1M sodium hydrogen

carbonate). The fluorescence generated was read at excitation 360nm and

emission of 450nm. This fluorescence is then compared to a standard by

developing a standard curve using a flurorescent standard as described

previously.


Enzyme assays were modified to develop mixing assays in order to determine

the inhibition of enzyme activity by patient serum. The assay was performed in

duplicate on the highest titre patient sample in a longitudinal series. A series of

two fold dilutions (starting from 1.25µg/ml) of recombinant human

Arylsulphatase B (Naglazyme) in dilution buffer (PBS, 0.05% Tween, 0.01%

HSA) were used to assess inhibition of enzyme activity. Ten microlitres of each

enzyme solution was then mixed with the same volume of patient sera and

normal sera in 96 well black plates to minimize the auto fluorescence and

background light scatter. The sera were added at fixed dilutions (1:4) and

incubated at room temperature for 1 hour. 4MU substrate (20 µl) in substrate

buffer (0.1M acetate buffer pH 5.4) was added to each well and incubated for 2

hours at room temperature in the dark. The enzyme activity in the absence and

presence of sera (for patient serum and normal serum) was measured. The

enzyme activity in the presence of patient serum was also compared with

enzyme activity of the plain enzyme solution for each dilution. Background

enzyme activity in patient serum and normal serum was measured.

82


as follows,


EANs- BEANs






This assay determines the inhibition of enzyme uptake by N-

acetylgalactosamine-4-sulphatase deficient fibroblasts in the presence of

antibodies. Fibroblasts from a MPS VI patient (MPS VI 94/097) were

established and maintained in DMEM, 10% FBS, 1% glutamine at 37oC and 5%

CO2. The assays were performed in duplicate. Six well culture plates were

seeded with 2.0x105 fibroblasts into each of the six wells. The cellular uptake

inhibition was performed once the wells were over 95% confluent, 24-48 hours

later. The enzyme (Naglazyme) was diluted in culture medium (DMEM with 1%

glutamine) at concentrations of 1.25µg/ml and 160ng/ml to ensure delivery of

enzyme into the cells. Patient serum or normal serum was added in a volume of

10 µl (1 in 100 dilutions) to the diluted enzyme solution (1000 µl volume) and

incubated for two hours at room temperature. The culture medium in the wells

was replaced with the enzyme mixed medium incubated with either normal or

patient serum. The cells were incubated for another two hours in 5% CO2 at

37oC. The cells were then washed with PBS and harvested by trypsinization.

The cells were resuspended, washed twice with PBS, and sonicated after a

cycle of freeze-thaw. After centrifugation at 2045G for 10 minutes, the

supernatant was tested for enzyme activity using 4MU assay (described earlier).

During the assay, the baseline arylsulphatase A and C activity of fibroblasts was

measured (prior to enzyme exposure) and subtracted from the final enzyme

83

activity (post Naglazyme exposure) to reveal the arylsulphatase B enzyme

delivered into the cells.

Cellular uptake inhibition (MPS VI)

Cell lysate(Enzyme)

Substrate

Patient serum

Enzyme in cell culture

medium

Serum

Incu

bate

A&C A &C

Aa

Patient serumNormal Serum

Normal serum

A&C

A&C

Figure 2.5 Cellular uptake inhibition in MPSVI

The protein concentration was determined by Bicinchoninic acid (BCA) assay

(Smith, Krohn et al. 1985). Arylsulfatase B activity was measured as follows,

Fibroblast arylsulphatase B activity = (Enzyme activity post Naglazyme

exposure) – (baseline Arylsulphatase A & C activity)

The per cent inhibition was calculated against plain enzyme solution as follows:

% Inhibition= 100-

100xEnzyme activity of lysate incubated in the presence of patient serum

Enzyme activity of lysate incubated with enzyme

84

2.6. MPS II

2.6.1. ELISA


solution (10µg/ml of elaprase in 0.1M NaHCO3, pH8.5) and stored at 4oC

overnight. After aspirating the excess enzyme next day the plate was washed

gently 5x with wash buffer (PBS, 0.1% Tween, pH7.4). 200ul of blocking Agent

(1% w/v HSA in 0.02M Tris /HCl, 0.25M NaCl buffer pH7.0) was added to each

well and plate stored at RT for 1 hr. The plates were then washed again with

wash buffer x5. Using dilution buffer (PBS, 0.05% Tween, 0.01% HAS), 2 fold

dilutions (16 dilutions in total) of specimens to be tested, a positive control and

negative control were prepared. To each well, 50µl of standard, control or

sample was added and plates were covered with an adhesive cover slip and

incubated at RT for 1 hr. After aspirating the tested samples the plates were

washed 5x with wash buffer. IgG goat anti human HRP antibody was added

(100µl) at a 1:5000 dilution to each well and incubated at RT for 1 hr. At the end

of incubation, the plates were washed 5x and 100µl of OPD was added to each

well. The plates were kept for 10 minutes at RT in a dark place. The reaction

was stopped by adding 50µl 2.5M H2SO4 and the absorbance of the plate was

read at 492nm immediately.

2.6.2. Enzyme assay in 96 well plates

Hunter syndrome (MPS II) results from a defect in the activity of the enzyme

iduronate-2-sulphatase (IDS). This fluorimetric assay for IDS uses 4-

methylumbelliferyl-α-iduronate 2-sulphate (MU-αIdoA-2S) as a substrate. To

exclude a dialysis step, lead acetate is added to the reaction mixture of crude

homogenate to precipitate phosphate and sulphate ions. Liberation of methyl

umbelliferone (MU) from MU-αIdoA-2S requires the sequential action of two

enzymes, desulphation by IDS followed by hydrolysis of MU-α-iduronide by α-

85

iduronidase. To avoid the second step being rate limiting, a second incubation in

the presence of additional α-iduronidase is undertaken. This exogenous enzyme

source of α-iduronidase also contains IDS activity but this is completely inhibited

by the high concentration of phosphate used in the second incubation.

Iduronate-2-sulphatase α-iduronidase

4-MU-αIdoA-2S 4-MU-αIdo

4-MU

The liberated 4-methylumbelliferone is measured using a luminescence

spectrophotometer against a standard of known concentration. The enzyme

activity assay was performed in duplicate. Specimens to be tested and artificial

substrate (MU-αIdoA-2S) prepared in substrate buffer (0.1M sodium acetate/

acetic acid buffer pH 5.0 containing 10mM lead acetate) were added to each

well of the plate in equal volumes (20µl). The plate was sealed with adhesive

film and incubated in a shaking incubator at 37oC for 4 hours. Phosphate citrate

buffer (double concentrated McIlvains phosphate / citrate buffer) was added (20

μl) to all samples and 10 μl purified lysosomal enzymes from bovine testis

(LEBT, available in the kit) was added and mixed in a shaking incubator at 37o

for 24 hours. At the end of the incubation, 140 µl of stop buffer (0.5 M

NaHCO3/0.5 M Na2CO3 buffer pH 10.7 + 0.025% Triton X-100) was added and

the fluorescence emission generated was measured using the microplate reader.

A 4-MU standard curve was prepared as described previously. The

fluorescence generated was read at excitation 360nm and emission of 450nm.

This fluorescence is then compared to a standard by developing a standard

curve using a flurorescent standard as described previously

86


The above enzyme activity assay was modified to develop catalytic inhibition

assay. The assay was performed in duplicate on the highest titre patient sample

in a longitudinal series. A series of two fold dilutions (starting from 5µg/ml) of

recombinant human iduronate-2-sulphatase (Elaprase) in dilution buffer (PBS,

0.05% Tween, 0.01% HSA) were used to assess inhibition of enzyme activity.

Ten microlitres of each enzyme solution was then mixed with the same volume

of patient sera and normal sera in 96 well black plates to minimize the auto

fluorescence and background light scatter. The sera were added at fixed

dilutions (1:4) and incubated at 37oC for 1 hour in a shaking incubator. The

enzyme activity of the mixture was then measured using 4MU substrate as

described earlier. This involved two additional incubation steps. The enzyme

activity in the absence and presence of sera (for patient serum and normal

serum) was measured. The enzyme activity in the presence of patient serum

was also compared with enzyme activity of the plain enzyme solution for each

dilution. The % inhibition of the catalytic activity for each enzyme dilution was

measured as follows,


EANs- BEANs





87

2.6.4. Cellular uptake inhibition assay

Due to a failure in developing a reliable mixing assay (catalytic inhibition) and

unavailability of a specific artificial substrate, development of cellular uptake

inhibition assay was not undertaken. This is discussed in more detail in chapter

3.

2.7. Western blot

The specificity of the IgG ELISA was confirmed by western blot analysis.

Proteins were separated using sodium dodecyl sulfate (SDS) polyacrylamide gel

electrophoresis (PAGE). This allowed various proteins to be separated based

on their molecular weight. Ten wells of SDS PAGE gel (10%) were prepared in

duplicate. Samples with protein concentration of 0.6μg per well were loaded in

the loading buffer. After loading enzymes and marker, an electric field was

applied (100 volt) until a ladder (marker) became apparent as proteins

separated (resolved) in the gel. At this point the electric current was increased

to 150 volts. The proteins were transferred to nitrocellulose membrane using the

semi dry transfer system (Constant 150 volt for 45 minutes). The membrane

was removed from the transfer system and blocking solution (5% milk in PBS-

Tween) was applied for 1 hour. The membrane was washed 3x in PBS (5

minutes per wash). The primary antibody (patient serum) was applied in a

dilution of 1:1000 and kept overnight at 4oC. Membrane was washed again 3x in

PBS-Tween (10 minutes per wash). The secondary antibody (anti human IgG

antibody) was applied at a dilution of 1:2500 for 1 hour at RT. The membrane

was washed again 3X in PBS-Tween (20 minutes per wash). Enhanced

chemiluminescence (ECL) solution was applied and photographic film was

prepared to image the antibody binding to the protein.

88

Figure 2.6 Western blot analysis

The figure describes the principle of western blot analysis. Proteins are separated, based on their molecular weight, by applying an electric current in a gel. These proteins are then transferred onto a membrane. Antibodies against a specific protein are identified by a ladder which marks the position of the protein on the nitrocellulose membrane.

2.8. Biomarkers and chimerism analysis

The ratio of DS to chondroitin sulphate (CS), a glycosaminoglycan (GAG) which

is not stored in cells in MPSI, is a better marker of disease progression than

urinary GAG (Church, Tylee et al. 2007; Langford-Smith, Arasaradnam et al.

2010; Langford-Smith, Mercer et al. 2010). We therefore, retrospectively

collected data on DS/CS ratio in our longitudinal patient series. Urinary GAG

and DS/CS ratio were performed in our clinical laboratory using two dimensional

electrophoresis (originally described by Whiteman (Whiteman 1973)) and

LabWorks software (Anachem, Luton, UK) (Church, Tylee et al. 2007). Analysis

of donor engraftment was performed by variable number tandem repeats

89

(VNTR) analysis using UVP gel photography system and LabWorks software

(Anachem Luton UK).

2.9. Statistics

Statistical analysis to compare multiple means of the same parameter was

performed by one-way analysis of variance (ANOVA) with the post-hoc Tukey’s

test. Analysis of data with more than two parameters was done by two-way

ANOVA via the standard least square method and post-hoc Tukey’s multiple

comparison. JMP software (SAS institute inc.) was used for these analyses.

Error bars represent standard deviation of mean and were calculated using

Microsoft Excel software.

90

3. Development of quantitative and

functional immune assays for Lysosomal

Storage Disorders

91

3.1. Introduction

To date, the majority of data on allo-immune responses to ERT have emerged

from the clinical trials conducted by the pharmaceutical companies developing

these therapies. These immune assays were developed, in order to comply with

the pharmaco vigilance guidelines, mandatory for the pharmaceutical industry.

These assays were based on highly sensitive immune antibody quantification

and detection technique i.e. ELISA (Mire-Sluis, Barrett et al. 2004; Shankar,

Shores et al. 2006). However, no clear strategy was developed by the

pharmaceutical industry, in order to study the functional effects of these

antibodies on delivered therapy. The multiplicity of antibody–enzyme

interactions means that a number of functional assays are required to

thoroughly investigate the effects of antibodies. Recombinant enzymes, in

clinical use, have a three dimensional structure with loci for interaction with

substrate, co-factors, cell surface receptors as well as other loci that are

redundant (Rempel, Clarke et al. 2005). Binding of antibody may block enzyme

activity (directly or indirectly), inhibit cellular enzyme uptake, change the cellular

compartment reached, enhance enzyme catabolism or may not have any

measurable impact at all.

Despite recent efforts to optimize and standardize these assays (Sellos-Moura,

Barzegar et al. 2011), these are not widely available and no clinically accredited

laboratories are currently offering these assays as a service. Development and

optimisation of these assays requires careful consideration of a number of

aspects of interaction between the antibodies and enzymes. It is important to

distinguish the binding and/or precipitation of the enzyme by the antibodies from

true catalytic or cellular uptake inhibition. Defining assay conditions is a major

challenge, and under suboptimum assay conditions, a strong inhibitory

specimen may not reveal true enzyme inhibition. In order to address this issue,

a series of experiments were undertaken to determine the optimum conditions

92

to elicit the functional nature of the antibodies. This required a number of

experiments performed under different conditions. A sensitive ELISA technique

was optimized to detect and quantify immune responses to ERT. Two different

types of functional assays were developed for MPSI, Pompe disease and

MPSVI. These included assays to evaluate inhibition of enzyme activity in vitro

by antibodies in patient serum (catalytic enzyme inhibition), and assays to

demonstrate the inhibition of enzyme uptake by patient fibroblasts which were

deficient in respective enzymes (cellular uptake inhibition).

3.2. MPSI

Aldurazyme is available as an ERT for patients with MPSI. The commercially

available product (Aldurazyme) was used in the experiments to develop

quantitative and functional assays.

3.2.1. ELISA

The first step in developing a sensitive IgG ELISA for Aldurzyme, is to define the

optimum concentration of enzyme to coat the EIA plates. During development of

ELISA, a negative cut off was defined for each dilution of serum. Specificity of

the assay was determined by simultaneously testing a serum with IgG anti-IDUA

antibodies and another serum sample with IgG anti-aglucosidase alpha

antibodies. Serum specimens from longitudinal patient series were tested to

assess sensitivity of the immune assay. The assay was repeated using EIA

plates prepared approximately 2 weeks prior to testing, to assess the stability of

plates.

A range of Aldurazyme concentrations were used to coat the plates and the

absorbance was measured after incubation with serum from patients with MPSI

treated with Aldurazyme as described in chapter 2. There was very little

absorbance detected on Aldurazyme concentrations of less than 1μg/ml. This

absorbance increased on higher enzyme concentrations and seemed to plateau

at 10 μg/ml and hence this concentration was selected as optimum Aldurazyme

93

concentration to coat the EIA plates as shown in figure 3.1. Serum from thirteen

normal individuals were used to define the mean negative control and a

negative cut off was defined as two standard deviations above the mean, in

order to include the 95% confidence interval for the value at a specific

concentration (figure3.2). The ELISA was repeated using a positive control

(MPSI patient with IgG antibodies) and another patient with Pompe disease who

had IgG anti aglucosidase alpha antibodies in serum. The ELISA was specific to

IDUA antibodies and the Pompe disease patient serum showed an absorbance

below the negative cut off consistent with a negative result (figure3.3). Serum

samples from an MPSIH patient over a period of time (longitudinal series) were

tested which showed the escalating immune response after exposure to

Aldurazyme peaking at a level of over 1:250000 dilutions which was the limit of

serum dilutions used for these assays (figure 3.4). ELISA plates showed the

same results when repeated 2 weeks or longer after preparation of plates (figure

3.5).

Effect of differing concentrations of Aldurazyme (µg/ml) EIA plate coating

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024

Ab

s 4

92

nm

Dilution Factor

20 10 5.00 2.50 1.25

0.63 0.31 0.16 0.08

Figure 3.1 Optimisation of EIA plate coating The figure shows absorbance of light as detected by microplate reader at a wavelength of 492nm. Serum dilutions ranged from 1:8 to 1:1024. Aldurazyme concentration ranging from 0.8 to 20μg/ml was used. The data shows that absorbance continues to increase till 10 μg/ml and then seems to plateau above that level.

94

Positive cut off

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8A

bs

49

2 n

m

Antibody titre

Specificity of assay

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Ab

s 4

92

nm

Dilution factor

MPS I

Negative control

Patient with anti rh-Aglucosidase alpha IgG antibody

Figure 3.2 Figure 3.3

Sensitivity of assay

1

10

100

1000

10000

100000

1000000

0 100 200 300 400 500 600

An

tib

od

y ti

tre

Days post ERT

ELISA

Stability of IDUA coated plates

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Ab

s 4

92

nm

Antibody titre

Plate Coated the previous evening

Plate coated 2 weeks prior to use

Figure 3.4 Figure 3.5 Figures 3.2-3.5 Optimisation of ELISA Figure 3.2 shows positive cut off (red line) for ELISA based on average absorption values from 13 different normal human sera (blue line). Please see text for details. Figure 3.3 shows negative ELISA using serum of a patient with IgG antibodies to aglucosidase alpha (green line) whilst the serum with anti IDUA antibodies is strongly positive (blue line).Figure 3.4 shows longitudinal ELISA data in a MPSI patient. Note positive ELISA at a dilution of 1:250000 on day 600 post ERT, confirming the highly sensitive nature of the assay. Figure 3.5 shows high absorbance of ELISA when tested in plates prepared two weeks earlier confirming the stability of coated plates over a period of time.

95

The specificity of ELISA was also demonstrated using a western blot analysis as

shown in figure. This figure shows that in the absence of antibodies in the pre

ERT sample, there is no antibody binding to Aldurazyme (recombinant IDUA) or

Myozyme (recombinant human alglucosidase alfa, Genzyme, Frmaingham,

USA). After exposure to ERT, the IgG antibodies in patient serum only bind to

Aldurazyme and not to Myozyme (figure 3.6).

Figure 3.6 Western blot analysis for MPSI Western blot for two patients depicting the specificity of ELISA for IDUA (Aldurazyme). In the absence of antibodies in the pre ERT sample (left panel), there is no antibody binding to IDUA or Myozyme (MZM). After exposure to ERT, the IgG antibodies in patient serum only bind to IDUA and not to MZM (right panel). Lower two panels are controls showing position of IDUA and MZM. Mkr (Marker) region shows the position of various bands based on their molecular weight (kDa) according to the ladder .

3.2.2. Catalytic enzyme assay

In clinical practice, enzyme activity assays are mostly performed in test tubes

which require large volume of substrates and patient samples. Due to

availability of only a limited volume of patient specimens, Aldurazyme enzyme

activity assays were optimized in 96 well plates. These assays were then

modified to develop catalytic inhibition assays. An inhibition of enzyme activity

was seen when normal serum was mixed with enzyme solution with known

enzyme activity. This response was seen with both normal serum and patient

serum. A number of experiments were undertaken to define the conditions

96

which would reveal the inhibition caused by antibodies in patient serum. This

included studying the effect of change in serum dilution, enzyme concentration,

pH, incubation times, using different dilution buffers and splitting the incubation

period.

The highest inhibition of enzyme activity was seen by using undiluted (neat)

serum. However, due to the availability of a limited amount of serum a dilution of

1:4 was used for the final analysis of patient specimens. The same inhibition

pattern was seen across the range of enzyme concentrations (15, 30 and

60ng/ml) as shown in figure 3.7. During this experiment an inhibition of over

25% was observed by both patient and normal serum at all enzyme

concentrations but there was no statistically significant difference between

normal serum and patient serum

97

0

2

4

6

8

10

12

14

16

No serum Neat serum

Dilution 1:2

Dilution 1:4

Dilution 1:8

4 M

U/h

ou

r

Aldurazyme 60ng/ml

MPS I serum

Normal serum

NS

NS

NS

NS

0

1

2

3

4

5

6

7

8

No serum Neat serum

Dilution 1:2

Dilution 1:4

Dilution 1:8

4 M

U/h

ou

r

Aldurazyme 30ng/ml

MPS I serum

Normal serum

NS

NSNSNS

00.20.40.60.8

11.21.41.61.8

No serum Neat serum

Dilution 1:2

Dilution 1:4

Dilution 1:8

4 M

U/h

ou

r

Aldurazyme 15ng/ml

MPS I serum

Normal serum

NSNSNS

NS

Figure 3.7 Effect of serum dilutions on catalytic inhibition

The figure shows inhibition of enzyme activity by neat serum and at three different dilutions (2, 4 and 8). Maximum inhibition was seen by neat specimens of patient and normal serum. This trend was observed in all three enzyme concentrations (15, 30 and 60ng/ml) used to assess the effect of serum dilutions. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. NS indicates no significant difference. Error bars refer to standard deviation of the mean

98

The IDUA enzyme assays for MPSI require an optimum acidic pH. In the

previous experiment, the pH of the buffer used for serum dilution was 7. Despite

the very small volume of dilution buffer (10μl), it could be argued that the use of

a dilution buffer at a higher pH could have caused some drop in enzyme activity.

In order to see the effect of change in pH, a dilution buffer with low pH (3.5) was

used which showed persistent inhibition by serum (figure 3.8) and there was no

statistically significant difference between dilution buffers at pH 7.0 when

compared to the dilution buffer at pH3.5. This confirmed that the inhibition seen

by the sera is due to the inhibitory effect of sera and not due to the altered pH.

Effect of change in PH

0

1

2

3

4

5

6

7

8

Normal Patient serum

4 M

U/h

ou

r

Enzyme activity Dilution 1:2 Dilution 1:4 Dilution 1:8

NS

No erum

pH 7.0 pH 3.5

No serum

Figure 3.8 Effect of change in pH

The figure shows the effect of change in pH. No significant difference in enzyme inhibition was observed at two different pH. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. NS indicates no significant difference. Error bars refer to standard deviation of the mean.

Early ELISA experiments had shown that the dilution buffer (buffer 2) used in

ELISA did not compromise the binding of antibodies to the enzyme coated

plates. During early catalytic inhibition assay, homogenizing buffer (buffer 1)

was used. Failure to demonstrate additional inhibition by antibodies in catalytic

inhibition could be due to denaturing of the antibodies during the process of

99

serum dilution. Therefore, inhibition assays were repeated using buffer 1 and

buffer 2 (figure 3.9). Dilution buffer 2 was the same buffer which was used in

ELISA experiments. Although these experiments showed significant inhibition of

enzyme activity, no significant difference was seen between the normal serum

and patient serum. Even though the difference between patient serum and

normal serum was not statistically significant, inhibition of enzyme activity by

patient serum was slightly higher than normal serum when buffer 2 was used

and hence buffer 2 was selected for subsequent experiments.

Effect of change in dilution buffers

0

1

2

3

4

5

6

7

8

Dilution buffer 1 Dilution buffer 2

4 M

U/h

ou

r

Enzyme (no serum) Patient serum Normal serum**

**** ****

NS

NS

Figure 3.9 Effect of change in dilution buffers

The figure shows the effect of change in dilution buffers. Significantly higher enzyme inhibition was seen using buffer 2. However the difference between normal serum and patient serum was not statistically different . Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. NS indicates no significant difference. Error bars refer to standard deviation of the mean.

100

Some antibodies require a longer incubation period to bind to the enzymes to

neutralize their effect. In order to evaluate the effect of incubation time, two

different incubation periods were compared to assess the enzyme inhibition.

These experiments showed that there was a statistically significant difference in

enzyme activities between each group. However, within each group the only

significant difference was between no serum to serum values (both patient and

normal serum). Again despite higher inhibition by patient serum, there was no

statistically significant difference between patient serum and normal serum. (see

(figure 3.10)

Effect of change in incubation time

0

1

2

3

4

5

6

30 minute incubation 2 hour incubation

4 M

U/

ho

ur

No serum Patient serum Normal serum

***

****

NS

Figure 3.10 Effect of change in incubation time

The figure shows the effect of change in incubation time. Reducing the incubation time to 30 minutes (figureb) results in loss of inhibition (compared to standard). Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. NS indicates no significant difference. Error bars refer to standard deviation of the mean

101

At this stage the incubation period was split into two incubations. The first

incubation period involved mixing of patient serum with enzyme solution and the

second incubation started after the substrate was added to the serum and

enzyme mixture. This experiment showed a statistically significant difference in

enzyme activity between baseline enzyme activity (no serum) and sera (patient

and normal serum). Also, there was statistically significant difference between

the patient serum and normal serum (figure 3.11).

Effect of two incubations

0

0.5

1

1.5

2

2.5

3

3.5

2 incubations

4 M

U/h

ou

r

Enzyme (no serum) Patient serum Normal serum

***

***

**

Figure 3.11 Inhibition using two separate incubations

The figure shows the effect of splitting the incubation period . There is significantly more inhibition by patient serum when compared to normal serum and no serum. Data was analysed by one-way ANOVA using post-hoc Tukey’s test. ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. Error bars refer to standard deviation of the mean

102

Splitting the incubation period, increasing the incubation time for first incubation

and using low enzyme concentration increased the difference between the

inhibition by patient serum and normal serum. As there was some inhibition

seen by normal serum, the enzyme activity in the presence of normal serum

was used as a standard to describe the inhibition caused by patient serum.

There was significant difference in enzyme activity across the enzyme

concentrations (p<0.01). See figure 3.12.

Two incubations across enzyme concentrations (rh-IDUA)

0

20

40

60

80

100

120

140

60 30 15 7.5 3.75 1.8 0.9 0.45

% E

nzy

me

acti

vity

Enzyme concentration (ng/ml)

% Enzyme activity compared to standard

% Enzyme activity in the absence of serum

**

Figure 3.12 Effect of change in enzyme concentrations

After optimizing the experiment conditions, the assay was performed across the Aldurazyme concentration (60ng/ml to 0.45 ng/ml) . The same inhibition pattern was observed across the enzyme concentrations. This effect becomes more marked at low enzyme concentration. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. ** indicates P value ≤ 0.01. Error bars refer to standard deviation of the mean

103

The experiment was repeated using human innate enzyme from HEK cells. A

similar pattern of inhibition was seen. Figure 3.13 shows that the enzyme

activity in the presence of serum drops to nearly 60% of the standard at a low

concentration of enzyme lysate (6.25 μg/20μl). In subsequent experiments,

serum with no antibodies showed no significant inhibition of enzyme activity

confirming the specificity of this response (figure 4.5).

Figure 3.13 Inhibition of innate enzyme (human embryonic kidney cells

lysate)

The experiment was repeated using human innate enzyme from human embryonic kidney (HEK) cells. Inhibition of innate enzyme becomes more marked when using lower concentrations of enzyme (6.25 and 12.5 μg/20μl of HEK cells protein).

104

In order to demonstrate that this inhibition was caused by antibodies in patient

serum, the experiment was repeated using a patient serum before HSCT (with

high titre of IgG antibodies) and the same patient serum after HSCT (with no

detectable IgG antibodies). The same assay was repeated using innate IDUA

mouse liver IDUA at different concentrations. An inhibition of above 20% was

demonstrated, which suggested cross reactivity of human antibodies with innate

enzymes from both humans and other species (figure 3.14).

Figure 3.14 Inhibition of innate enzyme (mouse IDUA enzyme)

Figure shows that mouse enzyme is inhibited by patient serum suggesting cross reactivity of antibodies to mouse IDUA.

Based on optimisation experiments, buffer 2 with two hour split incubation was

selected for final assays. A range of enzyme concentrations was chosen to

evaluate the catalytic inhibition (see chapter 2).

105


A suitable enzyme concentration for cellular uptake was determined by

exposing enzyme deficient fibroblasts to a range of enzyme concentrations.

Inhibition assay was then performed on selected concentrations to optimise the

uptake inhibition.

Fibroblasts were seeded in 6 well plates as described in chapter 2 and exposed

to increasing concentrations of Aldurazyme for a fixed period of time (one hour)

to ensure delivery of enzyme into the cells. Excellent levels of enzyme were

delivered into the cells using a wide range of enzyme concentrations (see figure

3.15).

Suitable enzyme concentration

0

20

40

60

80

100

120

140

160

180

6000 3000 1500 750 375 187.5 93.75

µm

ol 4

MU

/10

0µ

g/h

ou

r


Enzyme acitivity

Figure 3.15 Optimizing Aldurazyme concentration for uptake inhibition

assay

Fibroblasts from MPSIH patient were seeded in 6 well plates and exposed to a range of enzyme concentration (6000ng/ml to 93.75 ng/ml). Enzyme delivery into the cells was achieved at all concentrations.

Concentrations at 1/2 Km (Km: maximum enzyme delivered into the cells) and

below were selected for further experiments. This was to ensure that the

106

antibodies are not overcome by excessive enzyme in the incubation medium.

Enzyme activity in fibroblast lysate was measured at 93, 187, 375 and 750ng/ml

in the presence and absence of serum from the patient with high titre IgG

antibodies. This experiment was repeated using the serum from a normal

individual. The enzyme activity in the presence and absence of serum was

compared to quantify percentage inhibition. Unlike catalytic inhibition, no

inhibition of cellular uptake of enzyme was seen at any enzyme concentration

by normal serum. The difference between enzyme activity without serum and

patient serum increased with reducing enzyme concentrations. Statistically

significant difference (p<0.05) between the enzyme activities was seen between

no serum and patient serum groups at all enzyme concentrations (figure 3.16).

The highest enzyme inhibition was seen at enzyme concentration of 93ng/ml

which was selected as the optimum concentration for analysis of serum

specimens for final analysis (figure 3.17).

Optimization of enzyme uptake inhibition

0

50

100

150

200

250

750 375 187.5 93.75

µm

ol 4

MU

/10

0µ

g/h

ou

r


Enzyme activity (no serum)

Enzyme activity (patient serum)

**

**

*

***

3.16 Cellular uptake inhibition at different enzyme concentrations

Enzyme activity without serum and patient serum in MPSIH fibroblast lysate was assessed at four different Aldurazyme concentrations. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. Error bars refer to standard deviation of the mean.

107

Uptake inhibition (optimization of enzyme concentration)

0

20

40

60

80

100

750 375 187.5 93.75

% I

nh

ibit

ion

Enzyme concentration

MPS I patient**

**

***

NS

**

3.17 Percentage cellular uptake inhibition at different enzyme

concentrations

Inhibition of enzyme uptake by MPSIH fibroblasts was assessed at four different Aldurazyme concentrations. Maximum inhibition of enzyme uptake was observed at 93ng/ml. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. NS indicates no significant difference. Error bars refer to standard deviation of the mean

108

3.3. Pompe disease

Myozyme is licensed for use in Pompe disease patients. Myozyme was

purchased and used for developing the immune assays.

3.3.1. ELISA

Similar to MPSI, different enzyme concentrations were evaluated for

optimisation of ELISA for Pompe disease. A negative cut off was defined by

testing normal sera. Sensitivity and specificity of the assay were evaluated by

high titre antibody Pompe disease patient serum and serum from a patient with

anti-IDUA antibodies. Three different Myozyme concentrations were used (0.1,

0.5 and 1μg/ml) to coat EIA plates. A concentration of 1μg/ml was chosen as

the optimum concentration for coating EIA plates based on the absorbance

values of patient serum and normal control (figure 3.18).

Optimizing Myozyme concentration ELISA

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

Ab

s 4

92

nm

Dilution factor

1ug Patient 1ug Control0.5ug Patient 0.5ug Control0.1ug Patient 0.1ug Control

Figre 3.18 Optimizing Myozyme concentration for coating EIA

plates

Three different Myozyme concentrations were used to assess the absorbance during development of IgG ELISA. The highest absorbance was seen at 1μg/ml Myozyme concentration as shown in the figures. Error bars refer to standard deviation of the mean.

109

ELISA for MPSI, a normal cut off was defined as a value, 2 standard deviations

above the mean absorbance for that dilution (figure3.19). Positive results at a

serum dilution of 1:250000 demonstrate highly sensitive nature of the assay

developed (figure 3.20). The assay was performed using a Pompe disease

patient serum and a serum known to have IgG antibodies to Aldurazyme. The

results confirmed the specificity of assay (figure 3.21a). Specificity of assays

was also confirmed by western blot analysis as shown in (figure 3.21b)

Cut off

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

Ab

s 4

92

nm

Dilution Factor

Sensitivity of ELISA

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

Ab

s 4

92

nm

Dilution Factor

Patient sample 1 Patient sample 2 Cut off


Figure 3.19 and 3.20 Negative cut off and Sensitivity of ELISA

Negative cut off was defined as explained in the text. Figure 3.20 shows very high level of absorbance (positive at all 16 dilutions) in patient sample 2. Error bars refer to standard deviation of the mean.

110


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

Ab

s 4

92

nm

Dilution Factor

Pompe disease patient serumSerum with IgG anti IDUA antibodies

Western blot analysis (Pompe disease)

150 KDa100 KDa80 KDa60 KDa

NGZ NGZMZM MZM

Figure 3.21 Specificity of ELISA assay

High absorbance seen by Pompe disease patient. However a patient sample with IgG anti IDUA antibodies remains negative at all dilutions tested. Error bars refer to standard deviation of the mean. Western blot analysis (3.21b) shows binding of antibodies in patient serum to Naglazyme only. There is no antibody binding with Myozyme.

111


Enzyme activity assay for Myozyme was optimized in a 96 well plate (see figure

3.22) as described in chapter 2. The enzyme activity plateaued at levels above

25μg/ml depicting the highest limit of detection of enzyme activity. From this

data, suitable enzyme concentrations were selected to develop mixing assay to

study the catalytic enzyme inhibition. A number of parameters including

incubation time, enzyme concentration and serum dilutions were evaluated to

optimise the assay.

Optimizing enzyme assay in 96 well plate

0

5

10

15

20

25

30

35

100.0 50.0 25.0 12.5 6.3 3.1 1.6 0.8 0.4 0.2

4 M

U/3

0 m

in

Enzyme concentration (μg/ml)

Enzyme activity

Figure 3.22 Optimizing Myozyme activity assay in 96 well plate

Error bars refer to standard deviation of the mean

Based on understanding of catalytic inhibition, developed during optimisation

experiments in MPSI patients, only split incubations were used to develop the

enzyme inhibition assays in Pompe disease. An inhibition of enzyme activity

was seen both with normal serum and patient serum and the difference was

statistically significant as shown in figure 3.23. However, unlike MPSI no

significant difference was seen between normal serum and patient serum

112

despite using experimental conditions similar to those of MPSI. In previously

described experiments, de Vries et al. used a much longer incubation period (de

Vries et al. 2010) and hence the effect of varying incubation times was studied

in subsequent experiments. Incubation time was increased to 4 and 24 hours. A

significant inhibition compared to normal serum was only seen after 24 hour

incubation. The enzyme activity with patient serum was significantly lower than

no serum across the range of concentrations used in this experiment (p<0.05)

see figure 3.23.

The effect of varying serum dilutions on a range of enzyme concentrations was

tested as shown in the figure 3.24. More inhibition (direct and compared to a

standard) was seen by concentrated (1:4 dilution) serum and the difference

between patient serum and normal serum was statistically significant at this

dilution. However, on dilution of patient serum to 1:8 and 1:16, this inhibition is

lost. Due to the availability of a limited amount of patient samples and

demonstration of quantifiable inhibition at 1:4 serum dilution, neat serum was

not used for the assay.

113

0

5

10

15

20

12.5 6.3 3.1 1.6 0.8

4M

U/3

0 m

in

Enzyme concentrations (μg/ml)

Enzyme activity Patient serum Normal serumNo serum

*

**

***

***

*

**

***

***

0

5

10

15

20

12.5 6.3 3.1 1.6 0.8

4M

U/3

0 m

in



****

*****

****

***

******

***

***

0

5

10

15

20

12.5 6.3 3.1 1.6 0.8

4M

U/3

0 m

in



**

***

**

2 hour incubation

4 hour incubation

24 hour incubation


The figure shows catalytic enzyme inhibition at three d ifferent incubations at 2, 4 and 12 hours across a range of enzyme concentrations (12.5 to 0.8μg/ml). Optimum inhibition (compared to normal serum) is seen at 24 hour incubation. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001 . Error bars refer to standard deviation of the mean .

114

0

5

10

15

20

12.5 6.25 3.125 1.5625 0.78125

4 M

U/3

0 m

inu

te


Enzyme activity Patient serum Normal serum

Dilution 1:4

No serum ***

***

**

***

***

**

**

0

5

10

15

20

12.5 6.25 3.125 1.5625 0.781254 M

U/3

0 m

inu

tes



Dilution 1:8

No serum

***

*****

***

******

0

5

10

15

20

12.5 6.25 3.125 1.5625 0.781254 M

U/3

0 m

inu

tes



Dilution 1:16

No serum

*

*

***

******

***

Figure 3.24 Effect of serum dilutions on catalytic enzyme inhibition

The figure shows catalytic enzyme inhibition using serum dilutions of 1:4, 1:8 and 1:16. Maximum inhibition (compared to standard) is seen at a serum dilution of 1:4 across the range of enzyme concentrations (12.5 to 0.78μg/ml). Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. Error bars refer to standard deviation of the mean .

115

These experiments were performed at very high enzyme concentrations.

However, pharmacokinetic data on Myozyme suggests that the maximum

concentration of enzyme achieved after infusion is approximately 200μg/ml.

Myozyme has a half-life of about 2.75 hours [Myozyme product literature,

scientific discussion EMA 2006]. This suggests that the levels of Myozyme in

serum would drop to below 1μg/ml within 24 hours of infusion. This necessitates

assessment of catalytic inhibition at lower enzyme concentrations and hence a

wide range of enzyme concentrations (12.5μg/ml - 0.1μg/ml) was chosen to

study the catalytic inhibition in final assay protocol. During optimising

experiments, a different patient serum (with higher antibody titre) inhibited the

enzyme activity completely at a concentration of 0.1μg/ml whilst there was

detectable enzyme activity at this concentration with normal serum (100%

inhibition). See figure 3.25.

Based on these experiments, for the final assay protocol, prolonged incubation

period (24 hours) at a dilution of 1:4 was selected. The inhibition was tested

across a wide range of enzyme concentrations as described in chapter 2.

116

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0.8 0.4 0.2 0.1

4 M

U/

30

min

ute

s



**

**

***

No serum

*

*

*

0

20

40

60

80

100

0.8 0.4 0.2 0.1

% In

hib

itio

n


% inhibition (standard) % inhibition

*

*

*

Figure 3.25 Catalytic inhibition at low enzyme concentrations

Catalytic enzyme inhibition was assessed by lowering the Myozyme concentration. The optimum inhibition was seen at 0.1μg/ml Myozyme. Figure (a) shows the actual enzyme activity and activity in presence of patient and normal serum at four different dilutions. Figure (b) shows the percentage enzyme inhibition. Data was analysed by 2-way ANOVA (a) and one way ANOVA (b) using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. NS indicates no significant difference. Error bars refer to standard deviation of the mean .

A

B

117


Initial experiment to deliver Myozyme into the fibroblasts from the Pompe

disease patient showed no delivery of enzyme into the cells across the range of

enzyme concentrations. However, in subsequent experiments higher doses

were used and incubation times were increased. These experiments showed

that higher Myozyme concentrations were required to deliver the enzyme into

the cells compared to Aldurazyme in MPSI experiments. Also, the incubation

time had to be increased to achieve quantifiable levels of enzyme in the Pompe

disease patient fibroblasts (figure 3.26). Incubation times of both 6 and 24 hours

delivered satisfactory levels of enzyme into the fibroblasts and hence a 6 hour

incubation period was used in subsequent experiments.

Enzyme uptake by Pompe fibroblasts

0

1

2

3

4

5

6

7

8

1.56 3.13 6.25 12.50 25.00 50.00 100.00 200.00

μm

ol 4

MU

/30

m/1

00μ

g


1 hour incubation 6 hour incubation 24 hour incubation

Figure 3.26 Optimizing enzyme concentrations and incubation time for

cellular uptake inhibition assay

The figure shows enzyme activity of Pompe disease fibroblasts after exposure to a range of Myozyme concentrations (1.56-200μg/ml). No uptake was seen at any concentration when cells were incubated for one hour. Increasing the incubation time resulted in successful delivery of enzyme into the cells.

118

No significant enzyme uptake inhibition was seen by normal serum when tested

at two different enzyme concentrations (see figure3.27). However, there was

significantly low enzyme activity in the presence of patient serum when

compared to normal serum and no serum (p<0.05), both at 12.5μg/ml and 6.25

μg/ml. For final analysis, the percentage inhibition was calculated by comparing

the enzyme activity in the presence and absence of patient serum.

Cellular uptake inhibition

0

1

2

3

4

5

6

7

12.5μg/ml 6.25μg/ml

μm

ol 4

MU

/30

min

/10

0μ

g

Total enzyme activity Enzyme activity (Normal serum)Enzym activity (Patient serum)

***

***

**

*

Figure 3.27 Cellular uptake inhibition at two different enzyme

concentrations

Enzyme activity in the presence of patient serum is reduced to approximately 50% when compared to no serum and normal serum. No inhibition was seen by normal serum and hence percentage inhibition was measured by comparing enzyme activity in the presence of patient serum with that of the enzyme activity in the absence of serum. Data was analysed by one-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. Error bars refer to standard deviation of the mean.

119

Figure 3.28 shows the enzyme activity in fibroblast lysate in the presence and

absence of patient serum (with antibodies). Enzyme inhibition was seen only by

Pompe disease patient serum with IgG whilst no significant inhibition was seen

in the absence of an immune response.

Specificity of assay (Pompe disease)

0

1

2

3

4

5

6

7

Serum with antibodies Serum with no antibodies

Mo

l 4M

U/3

0m

in/1

00μ

g

Enzyme activity without serum Enzyme activity with serum

*

NS

Figure 3.28 Specificity of Cellular uptake assay

Data was analysed by one-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05. Error bars refer to standard deviation of the mean.

120

3.4. MPS VI

Commercially available product Naglazyme was used to develop immune

assays for MPSVI patients.

3.4.1. ELISA

Two different Naglazyme concentrations were used to optimize enzyme

concentration for coating EIA plates. Enzyme concentration of 10μg/ml showed

excellent absorbance values and hence this concentration was selected for

coating the plates (figure 3.29).

.

3.29 Optimizing Naglazyme concentration for EIA plates

Error bars refer to standard deviation of the mean.

121

A cut off was defined similarly to ELISA for MPSI and Pompe disease i.e. 2

standard deviation above the mean value of absorbance at a given

concentration. Specificity of assay was determined by quantifying immune

response in serum of a patient with MPS VI and serum with IgG antibodies to

Aldurazyme (figure 3.30).


0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Ab

s 4

92

nm

Dilution factor

Cut off

MPS VI patient

Serum with IgG anti IDUA antibodies

Figure 3.30 Specificity of ELISA

The figure shows positive ELISA when using serum from an MPSVI patient treated with Naglazyme. Absorbance value of serum with IgG anti IDUA antibodies remained below the threshold (negative result). Figure also shows a negative cut off for the MPS VI ELISA. Error bars refer to standard deviation of the mean. A western blot analysis shows binding of antibodies to Naglazyme only.

122

ELISA assay on a longitudinal series of samples from a patient with MPS VI

showed a positive result at the limit of dilution (1:250000) showing high

sensitivity of the assay (figure 3.31).

Sensitivity of assay

1

10

100

1000

10000

100000

1000000

0 100 200 300 400 500

An

tib

od

y ti

tre

Days post ERT

MPS VI patient

Figure 3.31 Sensitivity of ELISA

The figure shows results of a longitudinal sample series of an MPSVI patient. The highest response suggests a positive ELISA at a serum dilution of (1:250000). Error bars refer to standard deviation of the mean.

123


In order to develop mixing assays, Nagalzyme activity assay was optimized in

96 well black plates. To evaluate the sensitivity of substrate, two different

concentrations of artificial substrate (10mg/ml and 5 mg/ml) were used. Both

substrate concentrations resulted in highly sensitive assays. In view of difficulty

in dissolving 10mg of artificial substrate per millilitre of substrate buffer, only

5mg/ml was selected as the optimum substrate concentration for subsequent

assays (figure 3.32).

Optimization of Arysulphatase enzyme assay in 96 well plate

0

5

10

15

20

25

30

35

40

4M

U/2

ho

ur


5mg/ml substrate 10mg/ml substrate

Figure 3.32 Optimisation of Arylsulphatase enzyme assay in 96 well plate

using to different substrate concentrations.

No significant difference was seen between the two concentrations. 4MU depicting the enzyme activity tended to plateau at 40μg/ml suggesting the upper limit of detection of enzyme activity. Error bars refer to standard deviation of the mean.

However, unlike MPSI and Pompe disease, no inhibition of enzyme activity was

seen on mixing patient serum with enzyme concentrate (see figure 3.33). There

was no statistically significant difference between no serum (Enzyme activity),

patient serum and normal serum groups at any enzyme concentration tested

during this experiment.

124

Catalytic inhibition assay

0

0.5

1

1.5

2

2.5

1.25 0.63 0.31 0.16 0.08 0.04 0.02 0.01

4M

U/

2h

ou

r



Figure 3.33 Catalytic inhibition assay using a range of Naglazyme

concentrations (0.01 to 1.25 μg/ml)

Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. No significant difference was seen between the three groups (p>0.05) at all concentrations tested suggesting no enzyme inhibition either by patient serum or normal serum. There was an overall statistically significant concentration effect (p<0.01). Error bars refer to standard deviation of the mean.

The experiment was repeated across a range of enzyme concentrations using

different serum dilutions and incubation times (2, 4 and 24 hours) but no

underlying enzyme inhibition was demonstrated either by patient serum or

normal serum (see figures 3.34 and 3.35).

125

0.00.51.01.52.02.53.0

2.50 1.25 0.63 0.31 0.16 0.08 0.04

4 M

U/2

ho

urs



24 hour incubation

No serum

0.0

0.5

1.0

1.5

2.0

2.5

3.0

2.50 1.25 0.63 0.31 0.16 0.08 0.04

4M

U /

2 h

ou

rs



4 hour incubation

No serum

0.0

0.5

1.0

1.5

2.0

2.5

3.0

2.50 1.25 0.63 0.31 0.16 0.08 0.04

4M

U/2

ho

urs



2 hour incubation

No serum


The figure shows catalytic enzyme inhibit ion at three different incubation periods (2,4 and 24 hours). No statistically significant difference was seen between patient serum, normal serum and no serum groups at any concentration using different incubation periods. There is however, an overall statistically significant concentration effect (P<0.01) between all concentrations. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. Error bars refer to standard deviation of the mean .

126

Effect of serum dilution

0

0.5

1

1.5

2

2.5

3

3.5

4

Patient serum Normal serum

4M

U/

2 h

ou

r

Enzyme activity Neat serum Dilution 1:2

Dilution 1:4 Dilution 1:8

No serum

Figure 3.35 Effect of change in serum dilutions

No significant difference was seen in enzyme activity using different serum dilutions. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. Error bars refer to standard deviation of the mean .

127


A high level of enzyme was delivered into the MPS VI patient fibroblasts when

they were exposed to Naglazyme. Increasing the exposure time (incubation) to

six hours significantly enhanced the intracellular enzyme delivered into the cells

(figure 3.36). However, due to an adequate level of enzyme delivery into the

cells at one hour, this incubation period was selected to study the enzyme

uptake inhibition.

Enzyme uptake by MPS VI fibroblasts

0

5

10

15

20

25

30

35

40

45

50

2.5 1.25 0.625 0.3125 0.15625

4 M

U/1

00μ

g/ 2

ho

ur


1 hour incubation 6 hour incubation***

********

*

Figure 3.36 Optimizing Naglazyme concentration for cellular uptake assay

Excellent enzyme delivery was seen into the MPSVI fibroblasts at one hour incubation. Increasing the incubation time, significantly increased the uptake. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. Error bars refer to standard deviation of the mean.

128

Enzyme inhibition was tested at two different enzyme concentrations (1.25μg/ml

and 160ng/ml). However, no significant difference was seen between the

enzyme activity in the absence of serum and in the presence of patient serum

(with high titre antibodies), see figure 3.37. Increasing the concentration of

patient serum to 1:50 dilution also did not change the pattern of enzyme uptake

and no enzyme uptake inhibition was seen by either patient or normal serum

(figure 3.38).

Effect of enzyme concentration

0

5

10

15

20

25

30

35

40

45

160ng/ml 1.25mcg/ml

4 M

U/1

00μ

g/2

ho

ur

Naglazyme concentration

Normal serum Patient serum

Effect of serum dilution

0

5

10

15

20

25

30

35

40

45

Dilution 1:50 Dilution 1:100

4 M

U/1

00μ

g/2

ho

ur

Serum dilution



Figure 3.37 and 3.38 Cellular uptake inhibition assay at different

enzyme concentrations and serum dilutions

Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. No significant difference was seen between normal serum and patient serum at the two concentrations suggesting no inhibition. Also, no significant difference was seen using more concentrated serum (dilution 1:50). Error bars refer to standard deviation of the mean.

129

3.5. MPSII

Recombinant enzyme therapy (Elaprase) is available for clinical use in MPSII

patients. This product was purchased and used to develop immune assays for

MPSII patients treated with Elaprase.

3.5.1. ELISA

Two different Elaprase concentrations were used to optimize EIA coating and

10μg/ml Elaprase was selected as the optimum coating concentration (figure

3.39). A cut off was defined as two standard deviation above the mean value of

absorbance for a concentration. ELISA specificity was demonstrated by testing

MPS II patient serum and serum of MPSI patient with IgG antibodies to IDUA

(see figure 3.40).

Optimizing Elaprase concentration for EIA plates

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Ab

s 4

92

nm

Dilution factor

1 ug Patient 1 ug control

10 ug Patient 10 ug control

Figure 3.39 Optimzing Elaprase concentration for coating EIA plates

130

Specificity

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Ab

s 4

92

nm

Dilution factor

MPSII patient

Serum with IgG anti IDUA antibodiesCut off

Figure 3.40 Specificity of ELISA and negative cut off

Specificity of ELISA is demonstrated by a negative result when using a serum with IgG anti IDUA antibodies. A negative cut off is shown in the figure.


As described previously, the enzyme activity assay for Elaprase is a two step

process which involves sequential action of two enzymes. Desulphation of

artificial substrate by iduronate-2-sulphatase is followed by action of α-

iduronidase which releases 4MU. This enzyme activity assay was successfully

optimized in a 96 well plate (see figure 3.41), however, mixing assay required

addition of a third incubation period. The initial assays demonstrated no

significant inhibition of enzyme activity (figure 3.42) but repeat experiments on

highly complex assays (with three incubation periods) showed extremely high

inter assay variability as shown in figure 3.43 and 3.44.

131

Optimization of Iduronate-2-sulphatase assay in 96 well plate

05

101520253035404550

4M

U/

24

ho

ur


Enzyme activity

Figure 3.41 Optimizing iduronate-2-sulphatase enzyme activity assay in

96 well plate

Catalytic enzyme inhibition

0.00.20.40.60.81.01.21.41.61.82.0

4M

U/

24

ho

ur

Enzyme concentration μg/ml


******

***

*

******

***

****

******

No serum

Figure 3.42 Catalytic inhibition assay

The figure shows no inhibition of enzyme activity in the presence of normal serum and patient serum. A high enzyme activity is seen when patient serum or normal serum is mixed with enzyme solution at some dilutions. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. * indicates a P value ≤ 0.05, ** indicates P value ≤ 0.01, *** represents P value ≤ 0.001. Error bars refer to standard deviation of the mean.

132

Catalytic inhibition (2.5 μg/ml)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0.5 1.5 2.5 3.5

Enzy

me

act

ivit

y (4

MU

/ 2

4 h

ou

r)

Average

No serum Normal serum Patient serum

NS

NS

Catalytic inhibition (1.25 μg/ml)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.5 1.5 2.5 3.5

Enzy

me

act

ivit

y (4

MU

/ 2

4 h

ou

r)

Average

No serum Normal serum Patient serum

NS

NS

Figures 3.43 Figures 3.44

Figures 3.44 and 3.45 Catalytic inhibition at two enzyme concentrations

Variable enzyme activity was seen after inhibition assay as shown in the figures. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. Error bars refer to standard deviation of the mean.

133

3.6. Discussion

Even though the immune assays for different LSDs were based on the same

principle, the conditions of each experiment had to be changed considerably.

This highlights the different characteristics and variable nature of antibodies

produced in response to different ERTs. ELISAs for all LSDs were highly

sensitive and antibodies were still detected at extremely dilute serum samples of

over 1: 200,000 dilution. In contrast, catalytic inhibition assays were more

difficult to perform and required a combination of specific sets of conditions to

elicit the underlying inhibition by antibodies in patient serum. It is interesting to

note that in MPSI and Pompe disease, both the normal serum and patient

serum (with high titre antibodies) inhibit the enzyme activity. This could be due

to the inhibition by non-specific immunoglobulins and complement proteins in

normal serum. Therefore the enzyme activity of the enzyme-patient serum

mixture was compared with enzyme-normal serum mixture (standard) to reveal

the inhibition caused by antibodies in patient serum. Initial experiments for

catalytic inhibition assay, showed that there was no statistically significant

difference in enzyme activity when tested under different enzyme

concentrations, dilutions, incubation time, dilution buffers and pH. Increasing the

incubation time to 2 hours increased the inhibition by patient serum compared to

normal standard but this difference was not statistically significant. Likewise,

catalytic inhibition using buffer 2 as a dilution buffer, was higher than buffer 1

(p<0.01) and hence buffer 2 was adopted for subsequent assays. In the final

assay protocol, the incubation time was increased and two incubations were

used. The first incubation involved mixing the patient serum/normal serum with

enzyme at room temperature. At the end of this incubation, the substrate was

added to the mixture, followed by a second incubation. A wider range of enzyme

concentrations was used for final analysis of samples as outlined in the assay

protocol described in chapter 2. At low enzyme concentrations, the percentage

inhibition by patient serum in comparison to normal serum and no serum

appears high. Assessment of catalytic inhibition at low concentration is

important because the serum enzyme concentrations, unlike leucocyte enzyme

134

concentration, drop very rapidly after ERT infusion due to the short half-life and

rapid clearance of enzyme from the circulation. This is discussed in more detail

in chapter 4. The assays were repeated in the tolerized (no antibodies) and non-

tolerized (with high titre antibodies) serum, from the same patients, to confirm

that the inhibition against the standard (normal serum) was due to the

antibodies in the serum. In order to minimize background interference, baseline

enzyme activity in sera was measured during each assay and subtracted from

the final enzyme activity for patient and normal sera. A similar pattern of

inhibition was seen when innate human and mouse IDUA was used. This

phenomenon of cross reactivity of antibodies with innate enzymes of animal

origin has been described previously (Brooks, King et al. 1997). In contrast,

cellular uptake inhibition assays showed higher levels of inhibition at high

enzyme concentrations. It can be argued that the cellular uptake inhibition

assays are more meaningful because they incorporate two aspects of enzyme

inhibition. Firstly, they look at the ability of antibodies to block the enzyme from

getting into the cells which is a critical step required for the action of infused

exogenous enzyme. Secondly, due to the measurement of enzyme uptake

using a functional assay, one can only determine the catalytically active enzyme

taken up the cells. Hence these assays actually measure the uptake of

catalytically active enzyme, incorporating both aspects of functional assays.

Catalytic inhibition assays, in Pompe disease, required a much longer

incubation period of 24 hours. This necessitated carrying out a first incubation at

4oC. A different set of enzyme concentrations had to be used based on

optimisation experiments, in 96 well plates. Cellular uptake inhibition assay

required considerably higher enzyme concentration in 6 well plates seeded with

Pompe disease patient fibroblasts. However, increasing the enzyme

concentration was not enough to deliver the enzyme into the cells which is a

prerequisite to the measurement of cellular enzyme uptake inhibition. The

exposure time had to be increased to six hours. This finding is consistent with

the pharmacokinetic data on Myozyme (Amalfitano, Bengur et al.

2001)(Myozyme product literature). In clinical practice, the recommended dose

135

of Myozyme for Pompe disease patients is significantly higher than the dose of

Aldurazyme for MPSI patients. This poses an important question of whether the

current dosage strategies deliver adequate levels of enzyme into the enzyme

deficient cells of Pompe disease patients who develop high titre immune

response. Based on the data, it can be argued, that in the presence of high titre

immune response, either the dose of infusion or frequency of infusion need to

be increased to ensure delivery of therapeutic levels of Myozyme into the

patient fibroblasts.

MPS VI patients in contrast to MPSI and Pompe disease do not show evidence

of in vitro enzyme inhibition. This could be due to three reasons. It is possible

that, despite our experiments, we were unable to define the optimum conditions

to demonstrate enzyme inhibition. This will be supported by the fact that

previous experiments in animals and humans (White, Argento Martell et al.

2008) showed some evidence of in vitro enzyme inhibition. However, these

techniques did not involve the use of functional enzyme assays and did not look

at the true uptake of enzyme by the patient fibroblasts. A second possibility is

that the antibodies produced in MPSVI are not inhibitory as is shown in our

experiments. This is supported by the fact that there is no credible data showing

a correlation between the antibody titres and biomarkers of disease progression

or clinical deterioration. A third reason for a failure to show an inhibition could be

the relatively small number of patient samples in this group. We only tested

three patients and it is possible that none of them had inhibitory antibodies.

MPSII enzyme activity assay is a more complex assay which requires two

incubations. First step is desulphation, which is followed by IDUA exposure to

free 4MU from the artificial substrate. These steps are highly sensitive to time,

pH and temperature (Fluharty, Stevens et al. 1974; Mercelis, Van Elsen et al.

1979; Voznyi, Keulemans et al. 2001). This makes it considerably more difficult

to alter the experiment conditions to develop and optimize a catalytic inhibition

assay for MPSII patients using the currently available 4MU artificial substrate.

This was confirmed by initial experiments which gave inconsistent results during

the development of catalytic inhibition assay. Development of reproducible,

136

consistent and reliable functional assays for MPSII will require a different

substrate which is more specific, robust and does not require multiple incubation

steps.

137

Pulished in Haematologica 2012 Sep;97(9):1320-8 (appendix A)

4. Haemopoietic stem cell transplantation

improves the high incidence of

neutralizing allo-antibodies observed in

MPSI-Hurler after pharmacological enzyme

replacement therapy

138

4.1. Introduction

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-L-

iduronidase (IDUA). This results in intracellular accumulation of dermatan

sulphate (DS) and heparan sulphate (HS) and a progressive, multisystem

clinical disorder. Recombinant human ERT for LSDs first became available in

the 1990s (Barton, Brady et al. 1991) and is currently in clinical use for six LSDs

at considerable financial cost (Connock, Juarez-Garcia et al. 2006). ERT is

delivered into the enzyme deficient cells via M6P receptors. In order to

effectively clear the accumulated substrate inside the cells, the ERT has to be

metabolically active and penetrate the cells to reach the lysosomal

compartment.

An allo-immune response is widely reported to ERT and is summarized in table

1.4 (Ponder 2008). This phenomenon is not unique to ERT and antibodies are

reported in response to a number of infused recombinant human proteins.

(Porter 2001; Clarke, Wraith et al. 2009). Early data did not support the

functional nature and significant clinical impact of antibodies in MPSI (Kakkis,

Muenzer et al. 2001) but subsequently a prospective, open-labelled, multi-

national study revealed a suboptimal biomarker response in the presence of

high antibody titres (> 1:10,000) when compared to patients with no allo-immune

response (Wraith, Beck et al. 2007). More recent studies in canine models of

MPSI show a high level of enzyme uptake inhibition by anti IDUA antibodies

(Dickson, Peinovich et al. 2008). There is increasing evidence, mostly in animal

models of the LSDs, of an inverse correlation between an observed antibody

response and metabolic and clinical outcome. Even though the immune

response currently reported in patients with MPSI is 91%(Brooks, Kakavanos et

al. 2003), the true incidence of functionally active (neutralizing) antibodies is

unknown.

The consequences of persistent, functional allo-immune response can be

serious and range from reduced efficacy of ERT to catastrophic, rapid

progression of disease and high mortality (Shull, Kakkis et al. 1994; Wraith,

Beck et al. 2007; de Vries, van der Beek et al. 2010; Kishnani, Goldenberg et al.

139

2010). A number of immune tolerance induction regiments have been reported

in illnesses such as Haemophilia and MPSI (Kakkis, Lester et al. 2004).

Allogeneic HSCT can replace the enzyme naïve immune system with that of the

donor, thereby tolerizing the individual to the replaced enzyme. The donor

immune system is naturally tolerized to this protein the donor possesses normal

levels of this enzyme.

In children with severe MPSI (MPSI H, Hurler Syndrome) the standard of care is

allogeneic HSCT which is superior to ERT due to its ability to deliver enzyme

into the CNS. Pharmacological ERT is ineffective in neurocognitive disorders

and does not prevent neurological disease. In our centre, we deliver ERT to

patients with MPSI H prior to HSCT to improve the somatic (including cardiac)

manifestations of the disease and optimize the clinical status before potentially

toxic transplant conditioning (Wynn, Mercer et al. 2009). This enabled us to

collect blood samples from the recipients of MPSIH before and during the ERT

and HSCT.

We studied the incidence, pattern and impact of immune response in MPSIH

patients prior to receiving any treatment and following ERT and HSCT. We also

determined the relationship between these antibody responses and metabolic

biomarkers of the disease.

4.2. Patient demographics

The patients were categorized into two groups. One group included patients

who were followed longitudinally and comprised patients who received ERT

prior to HSCT (n=8). The second was a cross sectional group (n=20) of patients

all of whom were at least one year post HSCT. All patients in these two groups

had severe disease phenotype (MPSI H, Hurler Syndrome). One patient (patient

5) rejected the donor cells following HSCT and had autologous cells returned to

restore hematological function. This patient subsequently received a successful

second allogeneic HSCT. Longitudinal data for this patient during both

transplants are included in the results. Patient demographics, enzyme levels,

140

details of HSCT and immunosuppression for all patients in longitudinal series

and cross sectional series are described in table 4.1 and table 4.2 respectively.

No Gender Genotype Age at

HSCT (months)

Age at

ERT

(months)

Duration

of ERT (days)

Type of

HSCT

Source

of stem

cells

Conditioning

GVHD

Prophyl

axis

Enzyme

level at

diagnosis

Latest

Enzyme

post

HSCT

Donor

engraftment

(VNTR)

Complications

1 Male W402X/ W402X

6.9 4.2 83 MUD CB Bu, Flu, ATG CSA, Pred

0.22 55.5 100%

Autoimmune haemolysis; Rhinovirus

pneumonitis

2 Male W402X/ W402X

11.5 9.2 64 MUD CB Bu, Cy, ATG CSA, Pred

0.22 27.6 100%

VOD (late); skin GVHD; renal

tubular acidosis secondary to CSA

3 Male W402X/ W402X

12.2 8.5 96 MUD PBSC Bu, Cy,

Campath CSA, MTX

0.14 49.9 100% RSV pneumonitis; Pneumothoraces

4 Female Q70X/ W402X

8.3 4.5 114 MUD BM Bu, Cy,

Campath CSA 0.31 15.7 98%

Cy-induced acute cardiomyopathy

5 Male Q70X/ W402X

7.5 5.3 87 MUD CB Bu, Cy, ATG CSA, Pred

0.2 38.3 99%

Adenovirus; Gastrointestinal &

skin GVHD;Pneumonia

; Graft failure

6 Male R628X/ R628X

11.5 8.1 112 Sibling BM Bu, Cy,

Campath CSA 0.05 33.8 100%

VOD; CMV reactivation

7 Female W402X/Un-

known 13.2 10.2 113 MUD CB Bu, Cy, ATG

CSA, Pred

0.09 17.1 100% Norovirus

gastroenteritis

8 Male R628X/ R628X

5.0 0.9 124 Sibling BM Bu, Flu,

Campath CSA, MTX

0.06 20.1 70% VOD, adenovirus

infection

Table 4.1 Patient demographics (longitudinal patient series)

MUD; Matched unrelated donor, CB; Cord blood, BM; Bone marrow, PBSC; Peripheral blood stem cell, CSA; Ciclosporine A, Pred; Prednisolone, VNTR; Variable number tandem repeats, VOD; Veno occlusive disease, GVHD; Graft versus host disease, Bu; Busulfan, Cy; Cyclophosphamide, ATG; Antithymocyte globulin, RSV; Respiratory syncytial virus, Flu; Fludarabine, MTX; Methotrexate.

141

No. Gender Type of HSCT

Age at transplan

t (months)

Source of stem cells

Conditioning Immunosuppressi

on

Duration of immune

suppression (days)

Duion of ERT before HSCT

IgG Antibody Donor

engraftment (VNTR)

Complications

1 Male MUD 14.4 PBSC Bu, Cy, Campath CSA, Pred 154 120 Negative 100% GVHD (grade I), idiopathic

pneumonitis, CMV reactivation

2 Female MUD 11.0 CB Bu, Cy, ATG CSA, Pred 126 90 Negative 100% GVHD(grade I)

3 Female MUD 10.7 PBSC Bu, Cy, Campath CSA, MMF 122 72 Negative 100% GVHD(grade I)

4 Male MUD 15.4 CB Bu, Cy, ATG CSA, Pred 270 110 Negative 100% Chronic skin GVHD (grade I)

5 Female Sibling 11.0 BM Treo,flu CSA, MTX 90 150 Negative 100% EBV reactivation

6 Male Sibling 17.7 BM Bu, Cy CSA, MTX 105 90 Positive (1:256)

100% No significant problems

7 Male MUD 21.5 CB Bu, Fly, ATG CSA 122 95 Negative NA No significant problems

8 Male MUD 25.4 CB Bu, Cy, ATG CSA 229 240 Negative 100% EBV reactivation, Neutropenic

sepsis,, GVHD, VOD, Cardiomyopathy

9 Female MUD 15.3 BM Cy,Bu,Campath CSA, Pred 150 No prior ERT Negative 100% GVHD, Sepsis, cardiac arrest

10 Male Sibling 7.5 BM C,/Bu CSA 210 No prior ERT Negative 100% No significant problems

11 Male Sibling 31.3 BM Bu, Cy CSA, MTX 115 No prior ERT Weak positive

(1:128) 100% No significant problems

12 Male Sibling 10.8 BM Bu, Cy, ATG CSA, Pred 140 No prior ERT Weak positive

(1:64) 90% Neutropenic sepsis

13 Male Sibling 15.2 BM Bu, Cy, ATG CSA 188 No prior ERT Negative 90% GVHD skin (grade I), Neutropenic sepsis

14 Male Unrelated 16.6 CB Cy, Melphalan, ATG CSA 94 No prior ERT Negative 45% PTLPD, neutropenic sepsis

15 Male MUD 18.8 BM Cy, Melphalan, ATG CSA 85 No prior ERT Negative 100% CMV reactivation, Neutropenic

sepsis, EBV reactivation , PTLPD

16 Male Sibling 11.8 BM Bu, Cy, ATG CSA 154 No prior ERT Negative 70% Neutropenic sepsis, Gram

negative sepsis

17 Female MUD 22.7 BM Cy,Bu,ATG,Flu CSA 186 No prior ERT Negative 100% No significant problems

18 Female Sibling 13.4 BM Cy,Bu,ATG,Flu CSA, Pred 330 No prior ERT Negative 100% EBV reactivation, neutropenic sepsis, adenovirus infection, chronic limited skin GVHD

19 Male MUD 15.0 BM Bu, Cy, Campath CSA NA No prior ERT Negative NA No significant problems

20 Male MUD 14.9 PBSC Bu, Cy, flud,

Campath CSA 243 No prior ERT Negative 100%

GVHD (grade I), Gram negative sepsis, EBV reactivation

Table 4.2: Patient demographics (Cross sectional patient series) MUD; Matched unrelated donor, CB; Cord blood, BM; Bone marrow, PBSC; Peripheral blood stem cell, CSA; Cyclosporine A, Pred; Prednisolone, VNTR; Variable number tandem repeats, VOD; Veno occlusive disease, GVHD; Graft versus host disease, EBV; Ebstein-barr virus, CMV; Cytomegalovirus, Bu; Busulphan, PTLPD; Post transplant lymphoproliferative disorder, Cy; Cyclophosphamide, ATG; Antithymocyte globulin, Flu; Fludarabine, MTX; Methotrexate.

142

4.3. Pattern of immune response

4.3.1. ELISA

In the longitudinal patient series, high titres of IDUA reactive antibodies were

seen in 87.5% (n=7) patients during IgG ELISA. Figure 4.1 shows the immune

response after first exposure to ERT up until HSCT in these patients. One

patient (patient 8) demonstrated detectable IgG antibodies only at low titre

(1:256). These patients were followed up for a median period of 216 days

(range 155-685). The median time from first ERT exposure to a first positive IgG

antibody result was 38.5 days and the peak response 55.5 days (range 17-129).

Antibody titres in seven patients (immediate post-transplant data not available

for patient 6 in this group) who received HSCT had dropped to less than

1:10,000 by a median time of 101 days (range 26-137) despite having a normal

level of innate IDUA. Summary of immune response in all these patients is

shown in table 4.3.

Figure 4.1 Immune response (Pre-HSCT) in ERT treated MPSI patients

Longitudinal data describing the immune response in 8 patients (1 -8) in longitudinal series from start of ERT to just before HSCT is shown baselined to the time of first starting ERT. All patients raise antibody responses to enzyme.

143

Median time to first positive ELISA test 38.5 days (range 14-129)

Median time to highest immune response 55.5 days (range 17-129)

Median time to immune tolerance 101 days (range 26-137)

Incidence of high titre antibodies 87.5% (7/8)

Incidence of catalytic inhibition 62.5 (5/8)

Incidence of cellular uptake inhibition 75% (6/8)

Immune tolerance 1 Year post HSCT 100% (28/28)

Table 4.3. Summary of immune response in longitudinal patient series.

144

ELISA results from all patients over the entire follow up period are shown in

figure 4.2 base lined to the first HSCT. None of the patients had detectable

antibodies on the most recent ELISAs performed a year or more after the

HSCT. It is interesting to note that Patient 5 in this series had a primary graft

rejection with autologous hematological recovery after infusion of stored

autologous cells. This resulted in escalation of immune response following

subsequent exposure to IDUA which was later eradicated by a second

unrelated donor transplant.

Analysis of patient sera in cross sectional group (n=20) showed that 85%

(n=17) patients, tested one year or more after HSCT had no detectable

antibodies despite normal IDUA levels. ERT was given to 8 patients prior to

HSCT. Three patients in this group had weakly positive IgG antibody response

to IDUA (<1:250). Chimerism data was not available for two patients in this

group. The majority of patients (n=14, 78%) had achieved full donor

engraftment whilst 22% (n=4) were chimeric with donor leukocyte engraftment

of 45%, 70%, 90% and 90% respectively. Median duration of immune

suppression for Graft versus host disease (GVHD) prophylaxis after HSCT in

cross sectional and longitudinal patient series was 150 days (range 90-330) and

169 days (range 110-250) respectively (see table 5 and 6).

145

Figure 4.2 Immune response (Pre & post HSCT) in ERT treated MPSI

patients

Longitudinal data describing the immune response in 8 patients (1 -8) in longitudinal series is shown baselined to the time of first HSCT. Antibody titre is presented on primary vertical axis in logarithmic scale. Note a rapid decline in antibody titre after HSCT (following approximately 3 months of ERT). Patient 5 rejected his first graft which was followed by an esc alation of antibody titres. This immune response resolved after second transplant on day 328.

146

4.4. Enzyme neutralization by antibodies

Catalytic inhibition

Figures 4.3 describes the catalytic inhibition in all patients in longitudinal series

(direct enzyme inhibition and inhibition compared to a standard). In this series

62.5% of patients demonstrated catalytic inhibition. The inhibition was more

pronounced at lower enzyme concentrations. Three patients (patient 4, 5 and 8)

did not show enzyme inhibition at any concentration whereas another 2 patients

(patients 7 and 3) showed over 75% inhibition of enzyme activity at

concentrations of less than 1ng/ml. Direct inhibition of enzyme at higher

concentrations (30ng/ml or higher) ranged from 10-35% but inhibition in

comparison to a standard (Normal serum) was lower which is shown in figure

4.4. To confirm that the inhibition in patient serum was due to the antibodies, we

assessed the enzyme inhibition in the same patient before and after HSCT i.e.

specimen with the highest titre antibodies and no detectable antibodies on

ELISA. The data confirmed that there was no catalytic inhibition in the absence

of antibodies (figure 4.5).

147

Figure 4.3 Catalytic enzyme inhibition in MPSIH patients

Figure 4.4 Catalytic enzyme inhibition in MPSIH patients (compared to

standard)

Figures 4.3 & 4.4 Catalytic enzyme inhibition (Aldurazyme) in eight patients (1-8) and total inhibition in comparison to a standard (normal serum) across various enzyme concentrations.

148

Figure 4.5 Percentage inhibition in tolerized and non-tolerized serum

Inhibition due to tolerized and non-tolerized serum. Non-tolerized serum taken from a patient (patient 3) on ERT before HSCT with high titre antibodies is compared to tolerized serum taken from the same patient a year after HSCT (with no antibodies) which shows no significant inhibition .

***

149

4.4.1. Inhibition of cellular uptake of catalytically active enzyme

Cellular uptake inhibition was also assessed in the samples with the highest

antibody titre for each patient in the longitudinal series. Inhibition of enzyme

uptake by MPSI fibroblasts was seen in 75% (n=6) patients in this series. Figure

4.6 shows the uptake inhibition assay on these 8 patients. Two patients (patient

6 and 8) did not show any inhibition. It is interesting to note that one of these

patients (patient 6) had shown some enzyme inhibition on catalytic inhibition

assays. In contrast the two patients (patients 4 and 5) who did not show any

evidence of catalytic inhibition showed 25% and 76% inhibition of enzyme

uptake respectively. Patients 2 and 7 who showed over 75% inhibition of

enzyme activity also demonstrated a high level of cellular uptake inhibition (over

70%). There was no correlation between the catalytic inhibition and cellular

uptake inhibition in the rest of the patients suggesting a polyclonal nature of

antibodies. The cellular uptake inhibition was repeated in non-tolerized (patient

on ERT pre transplant and high titre immune response) and tolerized (post

HSCT with no antibodies) specimens from the same patient to confirm that the

inhibition was due to the antibodies in the serum (figure 4.7).

150

Figure 4.6 Cellular uptake inhibition in MPSIH patients

Cellular uptake inhibition in all patients (1-8) in longitudinal series. Patients 6 and 8 show no cellular uptake inhibition.

0

5

10

15

20

25

30

35

40

Non tolerized serum tolerized serum

μm

ol 4

MU

/100

mcg

/ho

ur

Specificity of assayEnzyme activity without serum Enzyme activity with serum

***NS

Figure 4.7 Cellular uptake inhibition in tolerized and non-tolerized MPSIH

patients

Tolerized serum (post-transplant with no antibodies) shows no inhibition compared to significant inhibition by non-tolerized serum (prior to transplant) in the same patient.

151

4.5. Immune response and disease biomarkers

Longitudinal biomarker data (DS/CS ratio) were available for six patients in this

series. Figure 7 describes the relationship between antibody titres and DS/CS

ratio over the follow up period. Patient 2 showed an immediate improvement in

the DS/CS ratio after starting ERT. This improvement in biomarkers was halted

by a high titre immune response. This pattern continued even after the HSCT

was performed and further improvement in DS/CS ratio resumed only after the

immune response was completely abrogated. DS/CS ratio in patient 3 stayed at

a high level despite HSCT, with a continuing high titre immune response. The

slight improvement in DS/CS ratio seen in this patient was suboptimal and could

be considered as a therapeutic failure at this stage. DS/CS ratio after resolution

of immune response is not yet available for this patient. The improvement in

biomarker levels in patient 4 was significant after ERT was commenced;

however, this progress was halted and partially reversed after a high titre

immune response was generated. The subsequent improvement in DS/CS ratio

followed the resolution of immune response. Patient 5 rejected his first HSCT

and had a second successful transplant. His ERT was interrupted during this

period. The initial improvement in biomarker levels plateaued during the high

titre immune response despite restarting ERT. After a second HSCT, biomarker

levels showed some deterioration despite ERT and HSCT which improved after

the immune response was completely resolved. Patient 6 showed a suboptimal

biomarker response to ERT during the high titre immune response. The DS/CS

ratio in patient 7 dropped significantly after ERT but further follow up data was

not available during the immune response and following the HSCT.

152




Figures 4.8-4.13 Correlation between biomarkers (DS/CS ratio) and the antibody titres in six patients in longitudinal series (patients 2-7). The shaded areas depict the period of ERT and black arrows point to the time of Stem Cell Transplant. 4.8) Patient 2 shows an immediate improvement in the DS/CS ratio after starting ERT which is halted by high titre anti IDUA antibodies. The biomarker levels

153

improve after the immune response is abrogated. 4.9) Patient 3 shows minimal improvement in biomarkers despite HSCT and ERT during high titre immune response. 4.10) The improvement in biomarker levels in patient 4 is significant after ERT is commenced; however, this progress is arrested and reversed after the high titre immune response is generated. The biomarkers improve after resolution of immune response. 4.11) Patient 5 rejected his first HSCT and had a second successful transplant. The initial improvement in biomarker levels plateau during the high titre immune response despite ERT (note a brief period when ERT was interrupted after first failed HSCT) which improves only after the immune response is completely resolved after second HSCT. 4.12) Patient 6 shows suboptimal response to ERT during the high titre immune response. 4.13) DS/CS ratio in patient 7 drops to a significant level but further follow up data is not available during the immune response and following the HSCT. Biomarker data is not available for patients 1 and 8.

154

4.6. Discussion

The immune response to replacement recombinant human proteins is widely

reported (Porter 2001). However the clinical impact of antibodies ranges from

no significant effect (Spijker, Poortman et al. 1982) to almost complete failure of

therapy (Yoshioka, Fukutake et al. 2003). In clinical disorders, such as

Hemophilia A and B, the immune response (inhibitory antibodies) can make the

replacement therapy (recombinant human factor VIII and IX) ineffective in some

cases. However availability of robust functional assays (Bethesda assays)

makes it easier to monitor the immune response and its impact in these

patients. In contrast, monitoring immune response in LSDs, is considerably

more challenging due to a lack of sensitive and standardised immune assays.

This is further complicated by the marked phenotypic heterogeneity that exists

in these disorders. It has been proposed that the allo-antibodies can neutralize

the infused enzyme by a number of mechanisms including altered enzyme

distribution, altered intracellular enzyme trafficking, increased enzyme turnover

and altered pharmacokinetic profile of infused enzyme (Brooks 1999). The

polyclonal nature of the IgG antibodies makes it imperative to develop multiple

functional assays to encompass the full range of effects due to antibodies (Mire-

Sluis, Barrett et al. 2004). Despite recent recognition of significance of

antibodies, no clinically accredited laboratories are performing functional

immune assays and hence the incidence and impact of these allo-antibodies

remains unknown.

Due to the difficulty in delivering ERT in some LSD patients with allergic

immune response and recognition of functional nature of antibodies, (de Vries,

van der Beek et al. 2010; Kishnani, Goldenberg et al. 2010) a number of

immune tolerance induction strategies were evaluated. These strategies had

variable outcomes and serious side effects were reported in some cases

(Brady, Murray et al. 1997; Hunley, Corzo et al. 2004; Kakkis, Lester et al.

2004; Joseph, Munroe et al. 2008). Intensification of immunomodulatory

therapies by using a combination of chemotherapeutic agents and

155

immunosuppressants could enhance the efficacy of these regimens but this

may result in a significantly increased morbidity as was seen in Haemophiliac

disorders treated with complex induction regimens (Berntorp, Astermark et al.

2000; Kreuz, Ettingshausen et al. 2003). HSCT has been used as a mean to

develop immune tolerance and control the symptoms in severe autoimmune

diseases. However, this modality has almost exclusively been used in

autoimmune disorders (Sullivan, Muraro et al. 2010). Manipulated graft, using

reduced toxicity conditioning can achieve immune tolerance induction in some

recipients of solid organ transplant (Kawai, Cosimi et al. 2008; Scandling,

Busque et al. 2008). However, despite all these developments and reduction in

transplant related mortality, HSCT as an immune tolerance induction

mechanism has not been used in allo-immune disorders.

This cohort included only MPSIH patients which is a severe phenotype. These

patients are expected to develop a higher titre immune response when

compared to more attenuated forms including MPSIHS and MPSIS. Seven

patients were homozygous for null mutation which confers a high risk of

developing allo-immune responses. Our results revealed a high incidence of

antibodies in this subgroup consistent with previous reports. All transplant

recipients, in the longitudinal series, cleared their antibodies to clinically

insignificant levels within a median period of 101 days despite normal enzyme

levels. None of the 20 patients in the cross sectional group had a high titre

immune response, one year or more after HSCT and cessation of

immunosuppression. Full donor engraftment was not required for immune

tolerance as some patients were tolerized despite achieving only mixed

chimerism. These data confirm allogeneic HSCT as an effective and quick

immune tolerance induction mechanism.

The majority of patients (62.5%) demonstrated evidence of some catalytic

inhibition by antibodies. Despite weak catalytic inhibition at high enzyme

concentration, this may reflect partial neutralization of infused enzyme due to a

156

very short half-life of Aldurazyme (3 hours in the absence of antibodies) and a

mean maximum plasma concentration (Cmax) of only 1.2-1.7μg/ml post infusion

[ALID-014-02: A phase II Open-Label Clinical Trial of Aldurazyme]. In previous

experiments, cross reactivity of these allo-antibodies was seen between

exogenous recombinant ERT and innate IDUA. These data show that the

antibodies generated in response to infused ERT can potentially neutralize the

innate enzyme produced by cellular ERT (allogeneic HSCT). This signifies a

need for HSCT induced immune tolerance.

In contrast to catalytic enzyme inhibition, cellular uptake inhibition experiments

were undertaken at a much higher enzyme concentration. Enzyme

concentration of less than ½ Km (100 ng/ml) and serum dilution of 1:100 were

selected during the optimisation experiments. Corrected for dilution, it will

equate to just over 6 times the Cmax (maximum plasma concentration post

infusion) of Aldurazyme. This shows that the treatment becomes virtually

ineffective in some patients with high titre immune response. The cellular

uptake inhibition at a higher enzyme concentration compared to catalytic

inhibition may reflect a stronger inhibition of the M6P binding sites (and enzyme

uptake) than the catalytic site of Aldurazyme by anti IDUA antibodies.

Analysis of biomarkers data and antibody titres show that the recovery is

slowed or in some cases reversed, in the presence of a high-titre immune

response. Generally there appears to be a close relationship between the two,

although DS/CS ratio and biomarker responses lagged behind antibody

responses usually by several days, confirming the in vivo neutralization of ERT

by antibodies.

These data show a high incidence of high titre functional immune response in

MPSIH patients. A close correlation between the biomarkers of disease and

antibody titres, confirm the in vivo effect of functionally active allo-antibodies.

157

These data also confirm allogeneic HSCT as an effective and quick immune

tolerance induction strategy.

158

5. The incidence and functional nature of allo-

antibodies in MPSI patients on long term

ERT

159

5.1. Introduction

Even though a high incidence of allo-immune response was reported in early

clinical trials in some LSDs, it was proposed that this response was transient in

nature due to seroconversion in some patients. A large multi-centre trial on

MPSI patients showed that despite generation of immune response in 90% of

patients on ERT, the majority of patients had cleared the immune response

within 6 months of treatment (Wraith, Clarke et al. 2004). A lack of sensitive and

widely available functional immune assays and an absence of clear correlation

between the presence of antibodies and clinical outcome increased the

uncertainty surrounding the significance of antibodies, particularly in the long

term recipients of ERT. However, persistent allergic reactions including

anaphylaxis were reported in patients receiving ERT on a long term basis

(Lipinski, Lipinski et al. 2009) suggesting that at least in some patients,

refractory immune response to delivered ERT persisted beyond 6 months and

remained problematic. Allergic reactions are mostly associated with IgE

antibodies whilst functional antibodies usually belong to IgG and IgM classes.

Nonetheless, generation and persistence of IgE antibodies, over a prolonged

period of time is a reflection of a dysregulated immune system and strongly

points towards a propensity in these individuals to develop persistent immune

response involving other antibody classes. It is important to remember that

unlike IgE mediated immune response which manifests with obvious clinical

features, allo-immune response involving other antibodies may remain

completely asymptomatic. Demonstration of the functionally active nature of

these antibodies means that the immune response can lead to neutralization of

delivered therapy even in relatively asymptomatic patients resulting in clinical

deterioration over a period of time (Banugaria, Patel et al. 2012). Neutralizing

allo-antibodies in long term recipients of ERT may have several implications

and hence it is critical to assess the immune response in patients receiving long

term ERT.

160

To address the question of whether functional allo-immune response persists in

long term recipients of ERT in MPSI, quantitative and functional immune assays

were performed in patients receiving ERT.

5.2. Patient demographics

Blood samples were collected from seventeen patients on long term

Aldurazyme therapy. Longitudinal data was available for 5 patients and 12 had

a single blood sample, taken at least 2 years after the start of treatment. The

median age at the start of ERT in this cohort was 4 years. The patients included

severe (n=2) and attenuated form of MPSI (n=15). Patient demographics and

genotypes are described in table 5.1.

No. Age at start

of ERT (years)

Sex Genotype Phenotype

1 2 Male L490P/L490P MPSI HS

2 8 Female C122del/unknown MPSI HS

3 3 Male Q380R/T388R MPSI HS


5 7 Female Q380R/ Q380R MPSI HS

6 7 Male P533R/P533R MPSI HS

7 1 Male NA MPS IH

8 5 Male A36E/Q70X MPSI HS

9 6 Female L490P/L490P MPSI HS

10 3 Female L490P/L490P MPSI HS


12 6 Female p.R628X/ p.R628X MPSI HS

13 1 Female W402X/W402X MPSI H


15 4 Male p.Gln380Arg/p.Thr388Arg MPSI HS

16 2 Male NA MPSI HS

17 0.5 Female L490P/L490P MPSI HS

Table 5.1 MPSI patient demographics (on long term ERT)

161

5.3. Allo-antibodies persist in some patients on long term ERT

The majority of patients (13/17) on long term ERT had IgG antibodies to

Aldurazyme. Seven patients had antibody titre of above 1:1000 and five were

positive at the highest limit of the assay. Median antibody titre at the time of last

assessment was 1:2048 in those who were positive. In longitudinal series

median time to immune response was 35 days and median time to maximum

immune response was 90 days. The patients in this series were followed for a

median period of 251 Days. All patients in longitudinal data were positive at the

time of last assessment as shown in figure 5.1. Two of the five patients in

longitudinal series had rising antibody titres at the time of last assessment. One

patient (patient 12) had an increase in antibody titre despite plateauing at a

level of 1:32000 approximately six months after starting the ERT. The summary

of immune response in all patients is shown in table 5.2.

162

Time Antibody titre

Catalytic inhibition

Uptake inhibition

1 >2 years Negative NA NA

2 >2 years 256 Yes No



5 >2 years 64 No No


7 >2 years 65500 Yes Yes

8 >2 years 320000 No Yes




12

0 day Negative

Yes

Yes

21 day Negative

35 day 32792

60 day 16336

83 day 32792

170 day 32792

600 day 262336

13

180 day 4096 No

Yes

210 days 4096

14

0 day Negative Yes

No

16 day Negative

30 day 262336

40 day 262336

15

90 day 32768 Yes

Yes

120 day 32768

251 day 2048

16 >2 years 131000 No Yes

17 0 day Negative Yes No

21 day 1024

Table 5.2 The summary of the immune response in MPSI patients (on long term ERT)

163

Longitudinal data ELISA (ERT only patients)

1

10

100

1000

10000

100000

1000000

0 200 400 600 800 1000

An

tib

od

y ti

tre

Days (post ERT)

Patient 12 Patient 13 Patient 14

Patient 15 Patient 17

Figure 5.1 Immune response in long term recipients of ERT (longitudinal

data).

The figure shows immune response in five patients over a period of time who were treated with ERT only. Patient 12 had a moderate sustained immune response between 3-6 months after exposure to ERT which seems to have escalated on most recent assessment, nearly two years after commencement of treatment. Patients 14 and 15 seem to have a declining immune response which remains above a titre 1:1000. Patients 13 and 17 have a relatively short follow up period and pattern of immune response is not fully established yet.

164

5.4. Catalytic inhibition

Of those, who had IgG antibodies on ELISA (n=13), 9 patients showed evidence

of direct catalytic inhibition (compared to no serum) ranging from 15-60%. A

similar pattern of enzyme inhibition was seen when compared to a standard.

Similar to the data described in chapter 4, these patients seemed to fall into

three categories when catalytic inhibition was assessed in comparison to a

standard at low enzyme concentrations as shown in figure 5.2. Four patients

(patients 4, 8, 13 and 16) did not show any significant inhibition. Five patients

(patients 7, 9, 12, 14 and 17) showed moderate catalytic inhibition ranging from

30-65% at low enzyme concentration (0.45ng/ml). All other patients lie in the

intermediate group and appear to show an inhibition of around 20% when

compared to a standard. Figure 5.3 shows direct inhibition of catalytic activity of

enzyme by patient sera. Similar to the data in chapter 4, direct enzyme

inhibition appeared slightly higher particularly at high enzyme concentrations

and there were no distinct groups at low enzyme concentrations. All patients

except patients 4,8,13 and 16 inhibited catalytic activity of enzyme.

165

% Enzyme inhibition compared to standard

0

20

40

60

80

100

30 15 7.5 3.75 1.8 0.9 0.45

% E

nzy

me

inh

ibit

ion


Patient 2 Patient 3 Patient 4 Patient 5Patient 7 Patient 8 Patient 9 Patient 12Patient 13 Patient 14 Patient 15 Patient 16Patient 17

Figure 5.2 Percentage catalytic enzyme inhibition compared to a

standard (normal serum)

% Enzyme inhibition

0

20

40

60

80

100

30 15 7.5 3.75 1.8 0.9 0.45

% E

nzy

me

inh

ibit

ion


Patient 2 Patient 3 Patient 4 Patient 5 Patient 7

Patient 8 Patient 9 Patient 12 Patient 13 Patient 14


Figure 5.3 Percentage catalytic inhibition (direct) in all patients with

positive immune response

166

5.5. Cellular uptake inhibition

Cellular uptake inhibition was assessed at a single enzyme concentration (93

ng/ml) as per established protocol. Six patients (35%) showed inhibition of

cellular enzyme uptake (see figure 5.4). Five patients demonstrated an

inhibition of over 60%. Three patients (patients 7, 8 and 16) in this group,

inhibited the uptake of Aldurazyme to over 90%. Six patients (patient 2,3,4,5 14)

showed no inhibition of enzyme uptake. All these patients except patient 4 had

shown some inhibition of catalytic activity as described previously. All patients

who inhibited the cellular enzyme uptake by over 90%, had generated a very

high titre immune response (>1:100000) on ELISA.

% Cellular uptake inhibition(ERT treated MPS I patients)

0

20

40

60

80

100

Uptake inhibition

Figure 5.4 Cellular uptake inhibition in all patients with positive immune

response.

167

5.6. Correlation between ELISA and catalytic inhibition

Correlation between ELISA and catalytic inhibition was assessed by Pearson

correlation coefficient using logarithmic values for both percentage catalytic

inhibition and ELISA. This analysis showed a poor correlation between the two

variables (correlation coefficient 0.18) as shown in figure 5.5.

Figure 5.5 Correlation between ELISA and cellular uptake inhbition

168

5.7. Correlation between ELISA and Cellular uptake inhibition

In constrast to catalytic inhibition, there appears to be a better correlation

between ELISA and cellular uptake inhibition (correlation coefficeint 0.49) as

shown in figure 5.6. Patient 14 could be identified as an outlier in this analysis.

Excluding this patient from analsyis, results in a correlation coefficient value of

0.67 suggeting a strong correlation between the ELISA and cellular uptake

inhibition. Spearman’s correlation coefficient value, for cellular uptake inhibition,

is calculated to be 0.84 which is suggestive of a very strong correlation between

these parameters.

Figure 5.6 Correlation between ELISA and cellular uptake inhbition

169

5.8. Discussion

Allo-immune response is widely reported to a number of recombinant human

proteins used in clinical practice. The severity, duration and functional nature of

immune responses are variable in different clinical disorders. A brief, transient

immune response may not have any clinical implications even if the antibodies

produced are inhibitory. On the other hand, a weak functional immune

response, which is sustained over a prolonged period of time, may cause

significant clinical deterioration. This makes it imperative to perform sensitive

detection assays followed by highly sensitive functional immune assays.

However, if an assay of low sensitivity is used to monitor the patients, there is a

risk that low titre immune response may not be detected despite potential

neutralization of infused protein by low level antibodies. Similarly, a high titre

immune response over a short period of time may not have any clinical

consequences for a particular patient. It is, therefore, important to monitor the

immune response regularly whilst patient is receiving ERT. The antibody titres

should be correlated with biochemical markers and clinical response. As

described previously, inferior metabolic response was reported in recipients of

ERT with high titre immune response (Wraith, Beck et al. 2007). Wynn et al. in

2009 reported metabolic outcome of the recipients of ERT and HSCT. In the

ERT group, outliers were reported who seemed to have suboptimal or minimal

immune response whilst on ERT (Wynn, Wraith et al. 2009). Similarly,

biomarker studies undertaken by our group showed unexpected fluctuations in

patients’ biomarker responses during ERT prior to HSCT (Langford-Smith,

Mercer et al. 2011). Some of these fluctuations may be explained based on the

generation of immune response which is functional in nature.

An important determinant of immune response, in recipients of recombinant

proteins, is the presence of CRIM. The patients with attenuated forms of

disease are less likely to have high titre immune response due to the presence

of residual protein which tolerises patients to the infused recombinant protein.

170

However, our data show that the majority of patients on long term ERT

developed immune responses regardless of their phenotype. In contrast to the

patients in chapter 4, the majority of patients in this cohort had an attenuated

phenotype. These data also show persistence of allo-immune response, beyond

six months, in the majority of patients with MPSI HS (11/15) who were treated

with ERT only. This makes it highly unlikely that the generation of immune

response is merely due to seroconversion. Patient 12 in longitudinal series had

persistent moderate immune response which escalated after a prolonged period

of ERT infusion. This is an important observation and suggests that

spontaneous tolerance does not always develop in patients with low or

moderate levels of immune response and all the patients on long term ERT

should have continuous monitoring of their immune response regardless of the

severity.

The data in chapter 4 and 5 suggest that the catalytic inhibition of Aldurazyme

does not depend upon the severity of immune response i.e. antibody titre. This

response appears to be idiosyncratic and becomes more obvious at low

enzyme concentrations. As explained in chapter 4, due to a low Cmax and short

half-life of Aldurazyme, inhibition at low enzyme concentration may reflect

significant inhibition of enzyme (in vivo) by circulating antibodies. However, it is

important to remember that the catalytic inhibition of circulating enzyme, outside

the cells, may not have any direct clinical consequences for the patient. The

enzyme has to be delivered into the cells to become effective which makes

cellular uptake inhibition more important. Nonetheless, the ability of antibodies

to inhibit the enzyme activity is an important indicator of functional nature of

these antibodies.

The data from cellular uptake inhibition assays show a high level of enzyme

uptake inhibition in some patients. The majority of patients who showed a high

level of enzyme uptake inhibition in this cohort and HSCT recipients (chapter 4)

had shown a high titre immune response on ELISA, suggesting that a high level

171

of antibodies is required to inhibit the uptake of enzyme into the fibroblasts. This

is in contrast to the catalytic inhibition assay, where the inhibition is more

unpredictable, and severity of catalytic inhibition does not depend upon the level

of antibodies in serum. This observation is supported by demonstration of a

better correlation between cellular uptake inhibition and ELISA titre when

compared to catalytic inhibition and ELISA titre (as measured by Pearson

correlation coefficient shown in figures 5.5 and 5.6).

The persistence of high titre, functionally active antibodies in patients on long

term ERT has several important implications. It highlights the significance of

monitoring quantitative and functional immune response in all recipients of ERT

regardless of their disease phenotype. The immune responders should then be

closely monitored in terms of biomarker and clinical response. In some patients

with attenuated disease type, neutralizing antibodies may make ERT ineffective

and hence alternative strategies including immune tolerance induction should

be considered. The presence of refractory immune response with deterioration

in biomarker and clinical status may warrant different therapeutic approaches

including HSCT. Continuation of a highly expensive therapy may not be

appropriate in these patients.

172

6. Allo immune response in other Lysosomal

Storage Disrders

173

6.1. Introduction

As described earlier, the problem of allo-immune response generation was

recognised in all LSDs. However the incidence, pattern and functional impact of

antibodies appear different in each one of these disorders. Most of the data on

the functional nature of antibodies comes from the animal studies and the

assays were performed in vitro (Brooks 1999). Unlike quantitative assays, the

development of functional immune assays is a great challenge and a number of

attempts have been made to develop reliable, robust and sensitive assays. The

reported incidence of allo-immune response in Pompe disease and MPSVI is

89% and 97% respectively. In Pompe disease the presence of antibodies have

been associated with poor clinical outcome (de Vries, van der Beek et al. ;

Abbott, Prater et al. 2011; Bali, Goldstein et al. 2012; Banugaria, Patel et al.

2012; Patel, Banugaria et al. 2012). Functionally active inhibitory antibodies

have previously been reported in some published studies (de Vries, van der

Beek et al. ; Patel, Banugaria et al. 2012). However, in MPSVI patients there is

generally a lack of compelling evidence about the inhibitory nature of allo-

antibodies. White et al. described functional assays to measure the

neutralization of enzyme by antibodies. Whilst enzyme neutralization in this

study was based on mixing assays, the quantification of true enzyme uptake

inhibition by patient fibroblasts was not performed (White, Argento Martell et al.

2008; White, Martell et al. 2008). The described assay used the antibody

binding to the calcium-independent mannose-6-phosphate receptor (CIMPR) as

a surrogate marker for the cellular enzyme uptake inhibition. This assay

provides indirect evidence of cellular uptake inhibition but does not reflect the

true uptake inhibition of enzyme by the antibodies. Similarly, there are

shortcomings in the functional assays developed by pharmaceutical industry

where normal human fibroblasts have been used instead of enzyme deficient

patient fibroblasts. The above two approaches may result in overestimation and

underestimation of enzyme inhibition respectively. A sensible approach towards

developing these assays would be to apply conditions, which closely mimic the

in vivo interaction of enzyme and antibodies with minimal manipulation of

174

patient samples and enzyme. As described previously, the allo-antibodies

generated in response to infused ERT are polyclonal in nature which means

that the same intensity of immune response (as measured by ELISA assay) in

different LSDs may not have same degree of inhibition as assessed by

functional assays. This could result in different clinical consequences for same

level of immune response in different LSDs. Similarly, within a disease group,

immune response in individuals with attenuated disease phenotype may not

have same implication as those with severe phenotype. However, recent data

suggest that in severe and attenuated forms of LSDs treated with ERT, the

presence of antibodies may be equally deleterious and result in adverse clinical

consequences (van der Ploeg, Clemens et al. 2010; Abbott, Prater et al. 2011;

Banugaria, Prater et al. 2011).

As described in chapter 3, the assays to evaluate the immune response and its

functional nature were developed and optimized for Pompe disease, MPS VI

and MPSII. An ELISA was performed on all patient samples followed by

quantification of catalytic enzyme activity inhibition and cellular uptake inhibition.

6.2. Pompe disease

6.2.1. ELISA

Six patients (10 samples) were tested for the presence of antibodies. Only two

patients were found to have IgG antibodies reactive to Myozyme on ELISA

assay. Patient 1 was positive at low titre (1:100) but became subsequently

negative on a repeat sample six months later during the course of treatment

with Myozyme. Patient 3 was found to have persistent high titre immune

response (>1:40000), over six months after the start of ERT. All other patients

were negative but it is important to note that some patients in this cohort had

received prophylactic immune tolerance induction treatment prior to

commencement of ERT. The specificity of ELISA was confirmed by western blot

analysis as shown in the figure 6.1.

175

No Time of

ELISA

ELISA

result

Age at

start of

ERT

CRIM

status Genotype

Clinical

presentation

Imm

un

om

o

du

lati

on

Patient 1 8 year 100 4 Positive NA Infantile No

8.5 year Negative

Patient 2 Pre ERT Negative 0.3 Positive

p.Trp746X/p.Trp746X

Infantile Yes

90 days Negative

180 days Negative

Patient 3 4 year 327680 10 Positive c.123IdelC/unknown Juvenile No

4.5 year 40960

Patient 4 3.5 year Negative 0.5 Positive NA Infantile No

Patient 5 1.5 year Negative 0.3 Negative c.525delT/p.Glu176ArgtsX45 Infantile Yes

Patient 6 4 year Negative 6 Positive g.1683G>T/ g.1683G>T Infantile No

Table 6.1 Patient demographics (Pompe disease)

Western blot analysis (Pompe disease)

MZMIDUA MZMIDUA

100 KDa80 KDa60 KDa

150 KDa

Figure 6.1 Western Blot analysis for Pompe disease

A western blot analysis for a patient showing the specificity of ELISA for alglucosidase alpha (Myozyme). Marker region shows the position of IDUA and Myozyme (MZM) bands based on their molecular weight (kDa) according to the ladder. After exposure to ERT, the IgG antibodies in patient serum only bind to MZM and not to IDUA.

176


Both patients with positive ELISA were tested for catalytic enzyme activity

inhibition assay. Patient 1 showed moderate inhibition at low enzyme

concentration and enzyme activity was completely blocked at a concentration of

100ng/ml Myozyme. Patient 3, however, showed a relatively low level of

enzyme activity inhibition when compared to a standard but remained inhibitory

throughout the range of concentrations of enzyme (see figures 6.2 and 6.3).

Catalytic inhibition assay was repeated using two samples from the same

patient. The sample with antibodies showed inhibition of enzyme activity but the

tolerized sample did not show any significant inhibition of enzyme activity when

compared to a standard suggesting that the inhibition in serum with antibodies

is due to the allo-antibodies to Myozyme (figure 6.4).

Catalytic inhibition (Pompe)

0

20

40

60

80

100

12.5 6.3 3.1 1.6 0.8 0.4 0.2 0.1

% In

hib

itio

n

Enzyme concentration (µg/ml)

Catalytic inhibition (patient 1)

% inhibition (standard) % inhibition

Catalytic inhibition (Pompe disease)

0

20

40

60

80

100

% In

hib

itio

n


Catalytic inihibition (patient 3)

% inhibition % inhibition (standard)

Figure 6.2 Figure 6.3 Figure 6.2 & 6.3 Catalytic enzyme inhibition in patients with Pompe disease Patient 1 showed complete inhibition of enzyme activity (direct and compared to a standard) at low enzyme concentration of 0.1Ug/ml. Patient 3 showed low level inhibition at all enzyme concentrations.

177

Specificity (Pompe disease)

0

10

20

30

40

50

12.5 6.3 3.1 1.6 0.8

% in

hib

itio

n



Serum with antibodies Serum with no antibodies

***

Figure 6.4 Specificity of Myozyme catalytic inhibition assay Pompe disease patient serum with IgG antibodies shows catalytic enzyme inhibition at all enzyme concentrations but the same patient serum (spontaneously tolerized) with no antibodies does not show any inhibition at any enzyme concentration. Data was analysed by 2-way ANOVA using post-hoc Tukey’s test. *** represents P value ≤ 0.001. Error bars refer to standard deviation of the mean.

178


Cellular uptake inhibition was performed in all samples which showed IgG

antibodies on ELISA. Patient 1 was negative and did not show any quantifiable

inhibition of enzyme uptake. Both samples (sample A and B) from patient 3

showed inhibition of cellular uptake of Myozyme. Sample A (high antibody titre)

showed above 40% inhibition of enzyme uptake at low (6.25μg/ml) and high

(12.5μg/ml) Myozyme concentration whereas sample B (low antibody titre)

showed lower uptake inhibition at high Myozyme concentration. The uptake

inhibition by sample B increased to 35% when performed at low enzyme

concentration (6.25μg/ml).

Cellular uptake inhibition(Pompe disease)

0

20

40

60

80

100

12.5μg/ml 6.25μg/ml

% in

hib

itio

n

Enzyme concentration

Cellular uptake inhibition

Patient 1 Patient 3 (sample A) Patient 3 (Sample B)

Figure 6.5 Cellular uptake inhibition Pompe disease

179

6.3. MPS VI

6.3.1. ELISA

All three patients tested for the presence of IgG antibodies against Nagalzyme

showed high titre immune response. Longitudinal samples were available for all

patients. Median time to development of immune response was 34 days.

Median time to highest immune response was 133 days. The patients were

followed for a median period of 503 days. All patients were positive at the time

of last assessment, a year or longer after the start of ERT (see figure 6.6).

Patient 1 had declining antibody levels at the time of last assessment whereas

patients 2 and 3 had stable antibody levels as shown in the figure. Patient 1

developed very high titre immune response during the course of ERT treatment

becoming positive at a serum dilution of over 1:250000 about four months after

commencement of therapy. Patient 2 and 3 remained moderately positive

during their treatment. The specificity of ELISA was confirmed by western blot

analysis as shown in figure 6.7.

180

Immune response in ERT treated MPSVI patients

1

10

100

1000

10000

100000

1000000

0 100 200 300 400 500 600

An

tib

od

y ti

tre

Days post ERT


Figure 6.6 Immune response in patients with MPS VI

Figure 6.7 Western blot analysis for Naglazyme

Western blot analysis shows binding of antibodies in patient serum to Naglazyme only. There is no antibody binding with Myozyme.

181


There was no direct inhibition of (Naglazyme) seen by any patient serum in this

cohort. When compared to a standard, only mild reduction in enzyme activity

was observed (about 10%) by patient 1 and 3 sera (figure 6.8 and 6.9). As there

were no MPS VI patient samples without any IgG antibodies (all samples

positive for ELISA), it was not possible to evaluate the specificity of this catalytic

inhibition.

Catalytic inhibition (MPS VI)

0

20

40

60

80

100

1.25 0.63 0.31 0.16 0.08 0.04 0.02

% in

hib

itio

n


Catalytic enzyme activity inhibition


Catalytic inhibition (MPS VI)

0

20

40

60

80

100

1.25 0.63 0.31 0.16 0.08 0.04 0.02

% in

hib

itio

n (

sta

nd

ard

)


Catalytic enzyme activity inhibition (standard)



Figures 6.8 & 6.9 Catalytic inhibition of enzyme (direct) and against a

standard.

182


All patients were tested to assess the inhibition of cellular uptake of enzyme at

two different enzyme concentrations (160ng/ml and 1.25μg/ml). No inhibition of

enzyme uptake was seen by any patient sera at both Naglazyme concentrations

as shown in the figures 6.10 and 6.11.


0

2

4

6

8

10

12

14

16

18

20


4M

U/1

00

µg

/2 h

ou

r

Cellular uptake inhibition (1.25 µg/ml)



0

5

10

15

20

25

30

35

40


4M

U /

10

0µ

g/2

ho

ur

Cellular uptake inhibition (160ng/ml)



Figures 6.10 & 6.11 MPS VI Cellular uptake inhibition assay at two

different enzyme concentrations

183

6.3.4. Correlation between antibody titre and biomarker

Longitudinal biomarker data were available for only one of the three patients as

shown in figure 6.12. A drop in DS/CS ratio was observed immediately after

commencement of ERT. However, despite very high titre antibody response, no

significant deterioration in biomarker levels was observed. Unfortunately,

biomarker data was not available for comparison in patients 2 and 3.

MPS VI (patient 1)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1

10

100

1000

10000

100000

1000000

0 100 200 300 400 500 600

An

tib

od

y ti

tre

Days post ERT

MPS VI patient DS/CS ratio

Figure 6.12 Correlation between antibody titres and Biomarkers in MPS VI

Longitudinal data showing allo-immune response and disease biomarker in MPS VI (patient 1). The biomarker level declines after the start of treatment and remains low despite persistent high titre immune response. There appears to be no correlation between the antibody titre and disease biomarkers in this patient.

184

6.4. Discussion

The reported incidence of allo-antibodies in Pompe disease and MPSVI is 89%

and 97% respectively. The significance of allo-immune response was

highlighted initially in CRIM negative patients where a poor clinical response to

ERT was attributed to development of immune response (Kishnani, Goldenberg

et al. 2010; Abbott, Prater et al. 2011; Bali, Goldstein et al. 2012). The

deteriorating clinical response in adult patients after generation of sustained

high titre antibody response (de Vries, van der Beek et al. 2010; Patel,

Banugaria et al. 2012; Regnery, Kornblum et al. 2012) highlights the

significance of immune response in attenuated forms of disease and those who

are CRIM positive. There is increasing evidence that the allo-immune response

in Pompe disease is detrimental and leads to clinical deterioration. In MPS VI,

despite very high incidence of allo-antibodies, the significance of antibodies

remains elusive. To date, there is no credible published case series which

shows a direct correlation between the allo-immune response and clinical

outcome in MPS VI patients treated with Naglazyme. This therapy seems to be

well tolerated despite development of persistent allo-immune response.

The incidence of antibodies in this cohort of Pompe disease patients is lower

than previously reported literature (2/7). The reported incidence of antibodies

(89%) in Pompe disease reflects development of antibody response at any time

after exposure of Myozyme, usually within first six months. Due to the

unavailability of longitudinal data for the majority of patients with Pompe

disease, it was not possible to evaluate the incidence of allo-antibodies post

Myozyme exposure. However, the two patients with positive immune response

confirm persistence of neutralizing allo-antibodies in long term recipients of

Myozyme. Our data show that both patients who developed immune response

to Myozyme had inhibitory antibodies. Catalytic inhibition did not appear to

correlate with the severity of immune response. Patient 1 with relatively low

titres of antibody inhibited the activity of Myozyme completely at low enzyme

concentration. Patient 2 had low level of catalytic inhibition despite high titre IgG

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immune response. In contrast, the cellular uptake inhibition appeared to be

dependent directly on the levels of antibodies present in the serum. The two

samples (sample A and B) of patient 3 had different levels of inhibition when

tested at two different enzyme concentrations. This could be due to excess

enzyme in high enzyme concentration medium, which the low titre antibody

serum was unable to neutralize. This uninhibited enzyme was then delivered

into the cells, despite the presence of antibodies. The second sample from the

same patient (with high titre antibodies) was able to inhibit not only the enzyme

at low concentration but also the medium with higher enzyme concentration.

All three MPSVI patients showed high titre IgG ELISA which is consistent with

previously published literature. It is very interesting to note that all MPSVI

samples, despite presence of a very high level of antibodies, did not inhibit

either catalytic activity of enzyme or its uptake by MPSVI fibroblasts. As

explained in chapter 3, this may reflect non-inhibitory nature of antibodies.

Previously, White et al. described functional inhibition by allo-antibodies in

patients with MPSVI who were treated with Naglazyme. However, in the

described assays, the actual uptake inhibition was not assessed and the assays

were not designed to quantify neutralizing activity of the allo-antibodies. In

contrast to the reported assays, our assays did not show any enzyme inhibition.

This could also be due to the less sensitive nature of our assays. However, due

to a small number of patients in this series it is difficult to conclude either way,

but the longitudinal biomarker data which were available for one patient in this

series did not correlate with antibody titres, supporting the notion that the

antibodies in MPSVI were not inhibitory in nature. At present, based on our

data, we can conclude that the antibodies generated in MPS VI patients do not

inhibit enzyme activity or cellular uptake and the titre of antibodies does not

correlate with biomarkers. This argument is also supported by a lack of credible

data which showing a clear correlation between antibodies and either biomarker

of disease progression or clinical outcome in MPSVI patients. On the other

hand, the presence of antibodies in patient serum may affect the enzyme

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activity by a different mechanism e.g. increased clearance of enzyme from

circulation. This would require a formal Naglazyme pharmacokinetic study in a

patient with high titre antibody. This mechanism of neutralization of enzyme

(rapid clearance) could, at least theoretically, be overcome by infusing a higher

dose of enzyme and would be influenced by other factors such as renal

function. In the absence of a formal PK study, it is difficult to comment any

further on the functional nature of allo-immune response in MPSVI.

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7. Conclusions and Future direction

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7.1. Aim1: Development of quantitative and functional immune assays

for LSDs

During this project, highly sensitive and specific quantitative immune assays

were developed which detected the presence of IgG antibodies in some patient

sera, diluted to a titre of over 1:200,000, confirming a high titre immune

response. Similarly, highly sensitive ELISA was developed to quantify immune

response for Pompe disease, MPSVI and MPSII. In our assays, unavailability of

human anti-enzyme antibody necessitated performing these assays with a

negative control and positive control for every sample (tested on 16 dilutions for

every sample). This makes these assays tedious, expensive and difficult to

standardise amongst different laboratories. Availability of human anti-enzyme

antibodies would make it possible to develop a single standard curve for a

number of specimens, enabling us to quantify antibodies in any given sample

regardless of the dilution. This will be an important step towards standardising

these assays. This in turn will lead to a reduction in inter laboratory assay

variation and make it easy to have a more robust internal and external quality

assessment mechanism.

Catalytic enzyme inhibition assays and cellular uptake inhibition assays,

developed during this project, were based on currently available protocols for

enzyme quantification assays for respective LSDs. Demonstration of catalytic

inhibition requires performing these assays across a range of enzyme dilutions

including extremely low enzyme concentration. At such concentrations, the

actual inhibition is subtle but in comparison to the standard or no serum, the

percentage difference becomes more remarkable and easy to quantify. The

inhibition at low levels of enzyme concentration is relevant because the

pharmacokinetic data available for various ERTs suggest that the serum

concentrations of enzyme drop to an almost undetectable level within a few

hours of ERT infusion. This makes it imperative to perform these assays at a

wide range of concentrations encompassing very low levels. However,

189

quantification of catalytic inhibition at different enzyme concentrations gives

different levels of inhibition at each of these concentrations which has relatively

limited clinical relevance and this information is unlikely to help clinicians in the

management of patients on ERT. None the less, this assay demonstrates the

functional nature of the antibody and hence forms an important part of the

evaluation of nature of antibodies. From a clinical perspective, however, it may

suffice to report the antibody as demonstrating catalytic inhibition rather than

the percentage inhibition across a range of enzyme concentrations. White et al.

developed catalytic assays by defining a cut off at certain enzyme concentration

for a normal serum (with no antibodies) (White, Argento Martell et al. 2008). Any

inhibition by patient serum (with antibodies) above this level was reported as

showing “catalytic inhibition” by tested serum. A similar method for reporting

could be adopted during further development, optimisation and validation of

these assays for clinical use in future.

In contrast to catalytic inhibition, cellular uptake inhibition assays are more

meaningful and provide clinically relevant information. The use of functional

enzyme assays to elicit cellular uptake inhibition means that these assays

actually demonstrate the cellular uptake inhibition of catalytically active enzyme

incorporating both aspects (catalytic and cellular uptake inhibition) of evaluation

of the functional nature of antibodies. Percentage inhibition and accurate

quantification of inhibition in this context is clinically relevant, as it provides

clinicians, with the basic information on the actual amount of enzyme delivered

into the cells at a given enzyme concentration.

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7.2. Aim 2: To study the incidence and impact of immune response in

MPSI patients treated with ERT and HSCT

The study of immune response in recipients of Aldurazyme, showed that the

majority of patients developed a high titre inhibitory immune response to

Aldurazyme. Our data showed allo-immune response in all MPSIH patients after

exposure to ERT. These data are consistent with previously published studies

which show a high incidence of immune response in recipients of Aldurazyme.

This immune response was subsequently abrogated by HSCT. However, it is

important to remember that all patients in this cohort had severe phenotype of

the disease i.e. MPSIH and were expected to have a higher incidence of allo-

immune response to ERT. Median time to development of immune response

was similar to the previously published data (Kakavanos, Turner et al. 2003).

Evaluation of immune response in recipients of ERT showed that the functional

immune response persisted in patients beyond six months, in some cases

inhibiting cellular uptake to nearly 95%. In view of our data (described in chapter

4), it would be reasonable to initiate a debate on developing a different strategy

for the management of patients who generate a neutralizing immune response

whilst on treatment with ERT. At present, management strategies particularly

the decision whether to offer allogeneic HSCT for patients with MPSI, are

predominantly based on the disease severity (phenotype and genotype).

Generally, allogeneic HSCT is only offered to patients with severe disease

phenotype i.e. MPSIH. Standard of care for attenuated disease type (MPSI

HS/MPSI S) is ERT. Whilst HSCT has been a well-established treatment for

MPSIH patients for decades, ERT is a relatively new treatment and its

limitations are not fully understood. Based on our data, it appears inevitable that

some patients, who develop allo-immune response to foreign proteins, will

generate a refractory response, making this therapy ineffective. Therefore,

when comparing the two treatment strategies i.e. HSCT and ERT, all factors

which could influence the outcome of treatment including refractory immune

response to ERT, need to be considered. A persistent refractory immune

191

response to ERT may warrant a different management approach such as

immune tolerance induction or HSCT. In this context allogeneic HSCT provides

a source of deficient enzyme as well as a mechanism of immune tolerance

induction. Significantly improved outcome in paediatric recipients of allogeneic

HSCT, makes this a viable therapeutic modality in attenuated type MPSI

patients who develop refractory neutralizing immune response.

7.3. Aim 3: To evaluate the nature and incidence of immune response in

other LSDs

Utilizing the assays, developed during this project, the functional nature of

immune response was studied in all available patient samples. The data,

confirms the presence of antibodies in all LSDs analysed, including MPSII,

MPSVI and Pompe disease patients. However, this project involved patients

from a single centre over a limited period of time and hence the numbers are

very small. Whilst significant inhibition was demonstrated by allo-antibodies to

Myozyme (in Pompe disease), no inhibition was seen by antibodies in MPSVI

patients (IgG antibodies against Naglazyme), despite high titre immune

response in all patients with MPSVI. These data may reflect a non-inhibitory

nature of antibodies in MPSVI. Previously, data from MPSI, showed that some

patients within an LSD group may not have inhibitory antibodies despite high

titre antibody response. In MPSI, the incidence of functionally active antibodies

was approximately 70% (as shown by the data in chapter 4 and 5). It is possible

that the incidence of functionally active antibodies in MPSVI is lower than MPSI

and none of the three patients tested in this study had functionally active

antibodies. This could be confirmed by testing more MPSVI patient samples.

However, this is an important finding and highlights the significance of

performing the functional enzyme assays in all recipients of ERT regardless of

the disease type, phenotype, disease severity and initial titre of immune

response. Collaboration between multiple centres involved in delivering the

ERT, could help evaluate the immune response in a larger patient series, which

will consolidate the data collected in this project and will provide further vital

192

information on the incidence and impact of immune response in these rare

disorders. Development of functional assays in some disorders e.g. MPSII may

not be straightforward, as explained in chapter 3, and may require development

of more specific substrates.

7.4. Perspectives and future experiments

In recent years, the focus of management in LSDs has shifted from

symptomatic treatment to disease eradication. Some of the available treatments

have a potential to cure the disease (HSCT and ERT) whilst others (SRT, CMT

and gene therapy) are either experimental or intended to arrest the disease

progression. During the last decade, ERT evolved into an established

therapeutic modality for a number of LSDs. However ERT is not universally

effective, has a very high cost burden and does not correct CNS disease due to

its inability to penetrate the BBB. Immune responses to ERT have recently been

recognized as a considerable clinical problem. In the past, a lack of

standardized assays and reliable biomarkers made it very difficult to assess the

therapeutic efficacy of ERT. Our data show that these antibodies may be

functionally active and correlate with biomarkers of disease progression in some

cases, resulting in reduced efficacy of this treatment. ERT puts a heavy cost

burden on health care systems. In the current climate with tightening of health

care budgets (Blumenthal and Dixon 2012; Greaves, Harris et al. 2012) it may

not be possible to continue delivery of very expensive treatments without

restriction. A logical strategy under these circumstances would be to identify the

patient groups which will show optimum response to the treatment.

Simultaneously, a prudent approach towards rationing the expensive treatments

e.g. ERT, is to identify the patients who will have limited or no response to

treatment soon after commencing the treatment. Such strategies to try and

identify the “therapeutic failures” will inevitably involve developing highly

sensitive and reliable biomarkers along with new tests which would predict the

193

therapeutic efficacy. This will not only help clinicians use resources more

effectively, but also refine management of the patients and plan alternative

treatments in case of therapeutic failure.

The problem of functional immune response generated against ERT can be

addressed by a number of different strategies. A change in structure of the

compound, rendering it less immunogenic could abrogate this problem

altogether. However, in view of the immunogenic potential of a replaced protein

(which is completely deficient in a recipient) and the complexity of human

immune response, this may not be possible in all individuals. Another strategy

that could be implied, may involve development of immune tolerance induction

regimens. The experimental data of immune induction in animal models of

LSDs is rather scanty and there is no general agreement on the optimum

induction methods. Various immune tolerance induction regimens have been

used in recent years which have variable efficacy with unsatisfactory results in

some patients. There are substantial risks associated with different immune

tolerance induction regimens depending upon their complexity and duration.

Development of optimum immune tolerance induction regimens is critical to

solving this problem. These strategies have been evaluated in animal models of

LSDs. A problem with current approach (Joseph, Munroe et al. 2008) of

studying the effects of immune tolerance induction in animals is the lack of a

good immune model which closely resembles the human immune system.

Published literature is based mostly on experiments in animals (Garman,

Munroe et al. 2004) including canine and murine models. However, these

models are far from perfect, and a number of novel human immune antibody

treatments available in clinical practice, cannot be used in these experiments to

evaluate their immune tolerance induction potential. Therefore, development of

human immune system is a prerequisite to developing an effective immune

tolerance strategy. A NOD/SCID/GammaC null mouse model can develop a

human immune system when injected with human CD34+ cells into the liver at a

neonatal stage (Ishikawa, Yasukawa et al. 2005; Siapati, Bigger et al. 2005).

194

These mice when injected with CD34 positive human haemopoietic stem cells

from an affected individual, can develop an enzyme naive human immune

system in mice. This model can then be used to study the in vivo effect of

antibodies on infused IDUA (Myozyme) and evaluate the novel tolerance

regimens e.g. human monoclonal anti CD20 and anti CD52 antibodies

(Rituximab, campath-1H) in a microenvironment very similar to humans. Other

neutralization mechanisms such as rate of clearance of enzyme from circulation

can also be performed by serial serum enzyme assays following IDUA infusion

in non-tolerized and tolerized mice. This kind of study will provide the crucial

data required to run a national or international human trial to explore and

optimize the management of this group of patients.

HSCT has a curative potential in patients with high titre immune response by

delivering cellular enzyme and inducing immune tolerance. High mortality and

significant morbidity make it a more intricate therapeutic option. Recent

developments in the field of HSCT have resulted in improved clinical outcome

and reduced transplant related mortality. This has made HSCT a viable

treatment option for a wider range of LSD patients. This can be used both as a

replacement therapy and immune tolerance induction strategy (saif, bigger et al

2012). Based on this evidence, it can be argued that failure of immune

tolerance induction should be considered as an indication for HSCT in certain

LSDs where the allogeneic HSCT is shown to be an effective therapeutic

modality. Due to the rapidity of disease progression in infantile LSDs with high

titre immune response, the decision to induce immune tolerance and offer

HSCT should be made promptly.

Inability of unmanipulated HSCT to correct disease pathology remains a

considerable problem for a number of LSDs e.g. MPSIII. Increasing the copy

numbers of delivered enzyme enhances the enzyme delivery and leads to

improved outcome in MPSI patients. In murine models, lentiviral vector-

mediated ex vivo HSC gene therapy has been shown to correct the disease

195

phenoytype (Biffi, De Palma et al. 2004; Biffi, Capotondo et al. 2006; Langford-

Smith, Wilkinson et al. 2012). Based on data from animal studies in MLD mouse

models, a clinical trial for MLD using lentiviral vector-mediated stem cell gene

therapy has been commenced. This clinical trial involves delivery of autologous

CD34+ cells transduced with the third generation lentiviral vector (hPGK.ARSA/

MOI=100/ double transduction) using a full intensity conditioning protocol. The

initial data from the trial shows excellent donor cell transduction and enzyme

levels nearly 13 times the normal levels (Prof. L. Naldini, ASGCT 2011, Dr. A.

Biffi, ESGCT 2011). In future, graft manipulation by gene therapy to deliver

supra-physiological enzyme levels, HSCT in early age and further decline in

transplant related mortality could open doors for extending the indications of

HSCT in other LSDs not yet amenable to this treatment

196

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Appendix A

Articles and Brief Reports Stem Cell Transplantation

*These authors contributedequally

Manuscript received onNovember 17, 2011. Revisedversion arrived on January 26,2012. Manuscript accepted on February 13, 2012.

Correspondence: Robert F. Wynn, Department ofBlood and Marrow Transplant,Royal Manchester Children’sHospital, Manchester, M13 9WL, UK.Phone: international+44.161.7018448. Fax: international+44.1617018410.E-mail: [email protected]

BackgroundMucopolysaccharidosis type I is caused by deficiency of a-L-iduronidase. Currently availabletreatment options include an allogeneic hematopoietic stem cell transplant and enzymereplacement therapy. Exogenous enzyme therapy appears promising but the benefits may beattenuated, at least in some patients, by the development of an immune response to the deliv-ered enzyme. The incidence and impact of allo-immune responses in these patients remainunknown.

Design and MethodsWe developed an immunoglobulin G enzyme-linked immunosorbent assay as well as in vitrocatalytic enzyme inhibition and cellular uptake inhibition assays and quantified enzyme inhi-bition by allo-antibodies. We determined the impact of these antibodies in eight patients whoreceived enzyme therapy before and during hematopoietic stem cell transplantation. In addi-tion, 20 patients who had previously received an allogeneic stem cell transplant were tested toevaluate this treatment as an immune tolerance induction mechanism.

ResultsHigh titer immune responses were seen in 87.5% (7/8) patients following exposure to a-L-iduronidase. These patients exhibited catalytic enzyme inhibition (5/8), uptake inhibition ofcatalytically active enzyme (6/8) or both (4/8). High antibody titers generally preceded eleva-tion of previously described biomarkers of disease progression. The median time to develop-ment of immune tolerance was 101 days (range, 26-137) after transplantation. All 20 patients,including those with mixed chimerism (22%), tested 1 year after transplantation were tolerizeddespite normal enzyme levels.

ConclusionsWe found a high incidence of neutralizing antibodies in patients with mucopolysaccharidosistype I treated with enzyme replacement therapy. We also found that allogeneic hematopoieticstem cell transplantation was an effective and rapid immune tolerance induction strategy.

Key words: immune tolerance induction, hematopoietic stem cell transplantation, Hurler’s syn-drome, mucopolysaccharidosis, enzyme replacement therapy, allo-antibodies.

Citation: Saif MA, Bigger BW, Brookes KE, Mercer J, Tylee KL, Church HJ, Bonney DK, Jones S,Wraith JE, and Wynn RF. Hematopoietic stem cell transplantation improves the high incidence of neu-tralizing allo-antibodies observed in Hurler’s syndrome after pharmacological enzyme replacementtherapy. Haematologica 2012;97(9):1320-1328. doi:10.3324/haematol.2011.058644

©2012 Ferrata Storti Foundation. This is an open-access paper.

Hematopoietic stem cell transplantation improves the high incidence of neutralizing allo-antibodies observed in Hurler’s syndrome after pharmacological enzyme replacement therapyMuhammad Ameer Saif,1,2* Brian W. Bigger,1,2* Karen E Brookes,1 Jean Mercer,3 Karen L. Tylee,3 Heather J. Church,3

Denise K. Bonney,2 Simon Jones,3 J. Ed Wraith,3 and Robert F. Wynn2

1Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, Manchester; 2Department ofBlood and Marrow Transplant, Royal Manchester Children’s Hospital, Manchester; and 3Genetic Medicine, St. Mary’s Hospital,Manchester, UK

ABSTRACT

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Introduction

Mucopolysaccharidosis type I (MPSI) is a lysosomalstorage disorder (LSD) caused by deficiency of α-L-iduronidase (IDUA). The deficiency of IDUA results inintracellular accumulation of dermatan sulphate (DS) andheparan sulphate and a progressive, multisystem clinicaldisorder. Recombinant human enzyme replacement ther-apy (ERT) for LSD first became available in the 1990s1 andis currently used clinically for MPSI, MPSII, MPSVI,Pompe, Gaucher and Fabry disease at a considerable finan-cial cost (estimated at $150,000-300,000 per patient perannum in MPSI).2 It is essential that the ERT is functionallyactive (catalytic activity) and able to target and penetrateenzyme-deficient cells. In MPSI, enzyme is taken up bycells via a mannose-6-phosphate receptor-mediated mech-anism. The formation of allo-antibodies has been reported to

occur in all LSD treated with ERT.3 The clinical importanceof antibodies in ERT-treated patients is uncertain but theexistence of an allo-immune response to infused exoge-nous enzyme and recombinant human proteins is welldescribed.4-5 In early clinical trials the reported incidence ofIDUA-specific antibodies was low (40%) and there wasno evidence of immunoprecipitation of enzyme or inhibi-tion of catalytic activity.6 Subsequently, a prospective,open-label, multinational study revealed a suboptimal bio-marker response in the presence of high antibody titers (>1:10,000) when compared to that in patients with no allo-immune response.7 More recent studies in canine modelsof MPSI demonstrated up to 90% inhibition of enzymeuptake by MPSI fibroblasts by serum taken from dogs thathad high titer anti-IDUA antibodies.8 There is increasingevidence, mostly derived from animal models of LSD, ofan inverse correlation between an observed antibodyresponse and metabolic and clinical outcome. Eventhough the immune response currently reported inpatients with MPSI is 91%,9 the true incidence of function-ally active (neutralizing) antibodies is unknown.The consequences of a refractory immune response can

be serious, ranging from treatment failure to catastrophicrapid progression of disease and high mortality in someLSD.7,10-12 Several clinical protocols for the induction ofimmune tolerance to exogenous enzyme have beenreported for these diseases and others, including hemo-philiac disorders13 in which there is a similar host immuneresponse against a foreign protein. Allogeneic hematopoi-etic stem cell transplantation (HSCT) can replace the recip-ient’s immune system with that of the donor, therebytolerizing the individual to the replaced enzyme (pharma-cological or cellular). The donor’s immune system is natu-rally tolerized to this protein as the donor possesses nor-mal levels of the enzyme. However, data on allogeneicHSCT as a means of immune tolerance induction arescarce.In children with severe MPSI (MPSI H, Hurler’s syn-

drome) the standard of care is allogeneic HSCT. Engrafteddonor leukocytes deliver enzyme (cellular ERT) to thebrain. Pharmacological ERT is unable to correct neurolog-ical disease because of its inability to cross the blood-brainbarrier. It has been our practice recently to give ERT topatients with MPSI H in the interval between diagnosisand transplantation in order to improve the somatic(including cardiac) manifestations of the disease and toprepare the child for transplant conditioning therapy.14 We

have developed quantitative and functional immunologi-cal assays and studied the incidence, pattern and impact ofthe immune response in MPSI patients prior to receivingany treatment and following ERT and HSCT. We havealso determined the relationship between these antibodyresponses and metabolic biomarkers of the disease.

Design and Methods

Patients’ samplesBlood samples were collected from 28 MPSI H patients treated

and followed up at the Royal Manchester Children’s Hospital overa 4-year period. The samples were collected with informed con-sent and under permission REC 08H101063 of the hospital’s ethi-cal commission. The patients’ blood samples were processedwithin 6 h and the serum was separated and stored at -80°C. Onegroup of patients was followed longitudinally and comprisedpatients who received ERT prior to HSCT (n=8). The second wasa cross-sectional group of patients (n=20) studied at least 1 yearafter HSCT. One patient (patient 5) rejected the donor cells follow-ing HSCT and had autologous cells returned to restore hematolog-ic function. This patient subsequently received a second allogeneicHSCT which was successful. Longitudinal data for this patientduring both transplants are included in the results. The demo-graphics, enzyme levels, details of HSCT, immune reconstitution,and immunosuppression for all patients in the longitudinal seriesare presented in Table 1. The transplant details and information onimmunosuppression for all patients in the cross-sectional series areshown in Table 2.

Immunoglobulin G enzyme-linked immunosorbent assayFor the immunoglobulin G (IgG) enzyme-linked immunosor-

bent assay (ELISA), 96-well plates were coated with recombinanthuman IDUA enzyme (Aldurazyme) (Genzyme, Framingham,USA) in enzyme-carbonate solution (10 mg/mL in 0.1 M sodiumbicarbonate, pH 8.5), overnight and blocked with blocking agentcontaining 1% human serum albumin. The plates were washedwith washing buffer [phosphate-buffered saline (PBS), 0.1%Tween at pH 7] three times and patient’s serum (50 mL) was added(two-fold serial dilutions) in duplicate and incubated at room tem-perature for 1 h. The plates were then washed three times withwashing buffer, and goat anti-human IgG-horseradish peroxidaseantibody (1:5000 dilutions in dilution buffer containing PBS,0.05% Tween, 0.01% human serum albumin) was added andincubated at room temperature for 1 h. Plates were washed again,o-phenylenediamine dihydrochloride solution was added and thereaction stopped with 2.5 M H2SO4. The plates were read at 492nm immediately. The normal range was determined for each dilu-tion by testing 13 normal sera (from normal volunteers). A positivecut-off value was defined as two standard deviations above theabsorbance value for the normal sera at that concentration. Toquantify ELISA, the lowest titer with absorbance above the cut-offwas reported as a positive serum dilution for that patient’s sample.Positive responses were confirmed by western blotting to showthe specificity of the IgG antibody to Aldurazyme.

Catalytic inhibitionFunctional enzyme activity was measured using 4-methylum-

belliferyl a-L-iduronide (4MU) substrate (Glycosynth, Warrington,UK) at 37°C based on previously described methods.15-16 We mod-ified the protocol to develop and optimize mixing assays in 96-well plates and assessed inhibition of enzyme activity by patients’sera. The assay was performed in duplicate on the highest titer

HSCT and immune tolerance

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Table 1. Patients’ demographics (longitudinal series of patients).N. Gender Genotype Age Age at Duration Type of Source Conditioning GVHD Duration CD3 CD19 Enzyme Latest Donor Complications

at HSCT ERT of ERT HSCT of stem prophylaxis of count at count at level enzyme engraftment(months) (months) (days) cells immuno- assessment assessment at level (VNTR)

suppression x106/L x106/L diagnosis post HSCT(days)

1 Male W402X/ 6.9 4.2 83 MUD CB Bu, Flu, CSA, Pred 184 330 733 0.22 55.5 100% AutoimmuneW402X ATG hemolysis,

rhinovirus pneumonitis

2 Male W402X/ 11.5 9.2 64 MUD CB Bu, Cy, CSA, Pred 250 1400 1470 0.22 27.6 100% VOD (late), W402X ATG skin GVHD,

renal tubular acidosis

secondary to CSA

3 Male W402X/ 12.2 8.5 96 MUD PBSC Bu, Cy, CSA, MTX 172 880 68 0.14 49.9 100% RSVW402X Campath pneumonitis,

pneumothoraces4 Female Q70X/ 8.3 4.5 114 MUD BM Bu, Cy, CSA 166 3994 666 0.31 15.7 98% Cy-induced

W402X Campath acute cardiomyopathy

5 Male Q70X/ 7.5 5.3 87 MUD CB Bu, Cy, CSA, Pred 110 370 160 0.2 38.3 99% Adenovirus,W402X ATG gastrointestinal

& skin GVHD,pneumonia,

6 Male R628X/ 11.5 8.1 112 Sibling BM Bu, Cy, CSA 165 1748 238 0.05 33.8 100% VOD,R628X Campath CMV reactivation

7 Female W402X/ 13.2 10.2 113 MUD CB Bu, Cy, CSA, Pred 184 854 794 0.09 17.1 100% Norovirus Un-known ATG gastroenteritis

8 Male R628X/ 5.0 0.9 124 Sibling BM Bu, Flu, CSA, MTX 155 1982 159 0.06 20.1 70% VOD, adenovirusR628X Campath infection

MUD: matched unrelated donor; CB: cord blood; BM: bone marrow; PBSC: peripheral blood stem cells; CSA: cyclosporine A; Pred: prednisolone; VNTR: variable number tandem repeats; VOD: veno occlu-sive disease; GVHD: graft-versus-host disease; CMV: cytomegalovirus; Bu: busulphan; Cy: cyclophosphamide; ATG: antithymocyte globulin; RSV: respiratory syncytial virus; Flu: fludarabine.

patient’s sample in the longitudinal series. A series of two-folddilutions (starting from 30 ng/mL to optimize the catalytic inhibi-tion) of Aldurazyme in dilution buffer (PBS, 0.05% Tween, 0.01%human serum albumin) were mixed with the same volume ofpatient’s sera or normal sera in serial dilutions in 96-well blackplates (Sterilin, South Wales, UK). The sera were added at fixeddilutions (1:4) and incubated at room temperature for 2 h in anincubator kept under agitation. The 4MU substrate (20 mL) in sub-strate buffer (0.4 M formate buffer, pH 3.5, 0.9% NaCl) was addedto each well and incubated for 1 h at room temperature in thedark. The enzyme activity in the absence and presence of sera (forpatient’s serum and normal serum) was measured. Normal serumwas used as a standard to compare and quantify the inhibitioncaused by the antibodies in the patients’ sera. The enzyme activityin the presence of a patient’s serum was compared with theenzyme activity of the plain enzyme solution for each dilution.We also used IDUA from human embryonic kidney cells (HEK)and mouse liver cells to assess the inhibition of innate enzyme bypatients’ sera.The percentage inhibition of the catalytic activity for each

enzyme dilution was determined as follows:

% Inhibition= 100-100x

where EAPs is the enzyme activity in the presence of thepatient’s serum, EANs is the enzyme activity in the presence of nor-mal serum, BEAPs is the background enzyme activity in the

patient’s serum and BEANs is the background enzyme activity innormal serum.

Cellular uptake inhibitionThis assay determines the inhibition of mannose-6-phosphate-

mediated enzyme uptake and subsequent activity by binding anti-bodies in non-FCR expressing, IDUA-deficient fibroblasts. MPSI fibroblasts (MPSI-A171) from a patient with Hurler’s

syndrome (genotype W402X/W402X) were established andmaintained in Dulbecco’s modified Eagle’s medium, 10% fetalbovine serum, 1% glutamine at 37°C in 5% CO2. The assayswere performed in duplicate. Six-well culture plates were seededwith 1.5×105 fibroblasts/well and grown to 95% confluence. Theenzyme was diluted in culture medium (Dulbecco’s modifiedEagle’s medium with 1% glutamine) to below half the Km forAldurazyme (100 ng/mL). Patient’s serum or normal serum wasadded at a volume of 10 mL (1 in 100 dilutions) to the dilutedenzyme solution (1000 mL volume) and incubated for 2 h at roomtemperature. The culture medium was replaced with the enzymemix medium and incubated for 1 h in 5% CO2 at 37°C. The cellswere washed with PBS and harvested by trypsinization. The cellswere re-suspended and washed twice with PBS and a final sus-pension was made in homogenizing buffer (0.5 M NaCl/0.02 MTris pH 7-7.5). Following a cycle of freezing and thawing, thecells were sonicated and centrifuged at 2045 g for 10 min. Thesupernatant was tested for enzyme activity using the 4MU assaydescribed earlier. The protein concentration was determined

EAPs- BEAPs

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Table 2. Patients’ demographics (cross-sectional series of patients).N. Gender Type Age at Source of Conditioning Immunosuppression Duration of Duration of IgG Donor Complications

of HSCT transplant stem cells immune ERT before antibody engraftment(months) suppression HSCT (VNTR)

(days)

1 Male MUD 14.4 PBSC Bu, Cy, Campath CSA, Pred 154 120 Negative 100% GVHD (grade I), idiopathic pneumonitis, CMV reactivation

2 Female MUD 11.0 CB Bu, Cy, ATG CSA, Pred 126 90 Negative 100% GVHD(grade I)3 Female MUD 10.7 PBSC Bu, Cy, Campath CSA, MMF 122 72 Negative 100% GVHD(grade I)4 Male MUD 15.4 CB Bu, Cy, ATG CSA, Pred 270 110 Negative 100% Chronic skin GVHD (grade I)5 Female Sibling 11.0 BM Treo, Flu CSA, MTX 90 150 Negative 100% EBV reactivation6 Male Sibling 17.7 BM Bu, Cy CSA, MTX 105 90 Positive 100% No significant problems

(1:256)7 Male MUD 21.5 CB Bu, Flu, ATG CSA 122 95 Negative NA No significant problems8 Male MUD 25.4 CB Bu, Cy, ATG CSA 229 240 Negative 100% EBV reactivation, neutropenic

sepsis, GVHD, VOD, cardiomyopathy9 Female MUD 15.3 BM Cy, Bu, Campath CSA, Pred 150 No prior ERT Negative 100% GVHD, Sepsis, cardiac arrest10 Male Sibling 7.5 BM C,/Bu CSA 210 No prior ERT Negative 100% No significant problems11 Male Sibling 31.3 BM Bu, Cy CSA, MTX 115 No prior ERT Weak 100% No significant problems

positive (1:128)12 Male Sibling 10.8 BM Bu, Cy, ATG CSA, Pred 140 No prior ERT Weak 90% Neutropenic sepsis

positive (1:64)13 Male Sibling 15.2 BM Bu, Cy, ATG CSA 188 No prior ERT Negative 90% GVHD skin (grade I),

neutropenic sepsis14 Male Unrelated 16.6 CB Cy, Melphalan, ATG CSA 94 No prior ERT Negative 45% PTLPD, neutropenic sepsis15 Male MUD 18.8 BM Cy, Melphalan, ATG CSA 85 No prior ERT Negative 100% CMV reactivation, neutropenic

sepsis, EBV reactivation , PTLPD16 Male Sibling 11.8 BM Bu, Cy, ATG CSA 154 No prior ERT Negative 70% Neutropenic sepsis,

Gram-negative sepsis17 Female MUD 22.7 BM Cy, Bu, ATG, Flu CSA 186 No prior ERT Negative 100% No significant problems18 Female Sibling 13.4 BM Cy, Bu, ATG, Flu CSA, Pred 330 No prior ERT Negative 100% EBV reactivation, neutropenic

sepsis, adenovirus infection, chroniclimited skin GVHD

19 Male MUD 15.0 BM Bu, Cy, Campath CSA NA No prior ERT Negative NA No significant problems20 Male MUD 14.9 PBSC Bu, Cy, Flu, Campath CSA 243 No prior ERT Negative 100% GVHD (grade I), Gram-negative

sepsis, EBV reactivation

MUD: matched unrelated donor; CB: cord blood; BM: bone marrow; PBSC: peripheral blood stem cells; CSA: cyclosporine A; Pred: prednisolone; VNTR: variable number tandem repeats; VOD: veno occlu-sive disease; GVHD: graft-versus-host disease; EBV: Ebstein-Varr virus; CMV: cytomegalovirus; Bu: busulphan; PTLPD: post-transplant lymphoproliferative disorder; Cy: cyclophosphamide; ATG: antithy-mocyte globulin; Flu: fludarabine; MTX: methotrexate.

using a bicinchoninic acid assay, as described elsewhere.17 Nouptake inhibition was observed with normal sera and hence thepercentage inhibition was calculated against plain enzyme solu-tion as follows:

% Inhibition= 100-100x

Biomarkers and chimerism analysisThe ratio of DS to chondroitin sulphate (CS), a glycosaminogly-

can (GAG) which is not stored in cells in MPSI, is a better markerof disease progression than total urinary GAG.18-21 We, therefore,studied DS/CS in our longitudinal series of patients. Total urinaryGAG and DS/CS ratio were determined using two dimensionalelectrophoresis (originally described by Whiteman22) andLabWorks software (Anachem, Luton, UK).19 Donor engraftmentwas evaluated in our clinical laboratory through analysis of vari-able number tandem repeats using a UVP gel photography systemand LabWorks software.

Results

Pattern of immune responseIn the longitudinal series of patients, high titers of IDUA

reactive antibodies were seen in 87.5% (n=7) of patientsduring IgG ELISA. Figure 1A shows the immune responseafter first exposure to ERT up to the time of HSCT in thesepatients. One patient (patient 8) had detectable IgG anti-bodies only at a low titer (1:256). These patients were fol-lowed up for a median period of 216 days (range, 155-685). The median time between first exposure to ERT andthe first positive IgG antibody result was 38.5 days andwhile that to the peak response was 55.5 days (range, 17-129). Antibody titers in seven patients (immediate post-transplant data were not available for patient 6 in thisgroup) who received HSCT had dropped to less than1:10,000 by a median time of 101 days (range, 26-137)despite these patients having a normal level of innate

Enzyme activity of lysate incubated in the presence of patient serumEnzyme activity of lysate incubated with enzyme



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IDUA. A summary of the immune responses in all thesepatients is shown in Table 3. The specificity of the ELISAwas demonstrated using western blotting, as shown inFigure 1B. This Figure shows that in the absence of anti-bodies in the pre-ERT sample, there is no antibody bind-ing to Aldurazyme (recombinant IDUA) or Myozyme(recombinant human alglucosidase-a, Genzyme,Framingham, USA). After exposure to ERT, the IgG anti-bodies in patients’ sera only bound to Aldurazyme and notto Myozyme. ELISA results from all patients over theentire follow-up period are shown in Figure 1C base-linedto the first HSCT. None of the patients had detectableantibodies on the most recent ELISA performed a year ormore after the HSCT. It is interesting to note that patient5 in this series had primary graft rejection with autologoushematologic recovery after infusion of stored autologouscells. This resulted in an escalation of the immuneresponse following subsequent exposure to IDUA whichwas later eradicated by a second unrelated donor trans-plant. In this cohort, the median CD3 and CD19 countswere 1140×106/L (range, 330×106/L - 3994×106/L) and452×106/L (range, 68×106/L – 1470×106/L), respectively, atthe time of the last assessment.Analysis of the sera from patients in the cross-sectional

group (n=20) showed that 85% (n=17) of patients tested 1year or more after HSCT had no detectable antibodiesdespite normal IDUA levels. Eight of these patients hadreceived ERT prior to HSCT. Three patients in this group

had weakly positive IgG antibody responses to IDUA(<1:250). Chimerism data were not available for twopatients in this group. The majority of patients (n=14,78%) had achieved full donor engraftment while 22%(n=4) were chimeric with donor leukocyte engraftment of45%, 70%, 90% and 90%. The median duration ofimmune suppression for graft-versus-host disease prophy-laxis after HSCT in the cross-sectional and longitudinalseries of patients was 150 days (range, 90-330) and 169days (range, 110-250), respectively (see Tables 1 and 2).The median time to assessment in this cohort was 66months (range, 12-181).

Enzyme neutralization by antibodiesFigure 2A illustrates the catalytic inhibition in all

patients in the longitudinal series. In this series 62.5% ofpatients demonstrated catalytic inhibition. The inhibitionwas more pronounced at lower enzyme concentrations.Three patients (patients 4, 5 and 8) did not show enzymeinhibition at any concentration whereas another twopatients (patients 7 and 3) showed over 75% inhibition ofenzyme activity at concentrations of less than 1 ng/mL.Enzyme inhibition at higher concentrations (7.5-30ng/mL) was up to 20% in comparison to that of a standard(normal serum). A similar pattern of inhibition was seenby antibodies when human and mouse enzymes wereused (data not shown).To confirm that the inhibition in a patient’s serum was

M.A. Saif et al.


Figure 1. (A) Longitudinal datademonstrating the immuneresponse in eight patients (1-8)in the longitudinal series fromstart of ERT to just before HSCTare shown baselined to the timeof first starting ERT. All patientsraised antibody responses tothe enzyme. (B) A western blotfor two patients depicting thespecificity of the ELISA for IDUA(Aldurazyme). In the absence ofantibodies in the pre-ERT sam-ple, there was no antibody bind-ing to IDUA or Myozyme (MZM).After exposure to ERT, the IgGantibodies in the patients’serum only bound to IDUA andnot to MZM. Mkr (Marker)region shows the position of var-ious bands based on theirmolecular weight (kDa) accord-ing to the ladder. (C)Longitudinal data demonstrat-ing the immune response ineight patients (1-8) in the longi-tudinal series are shown base-lined to the time of first HSCT.Antibody titer is presented onthe primary vertical axis in alogarithmic scale. Note a rapiddecline in antibody titer afterHSCT (following approximately 3months of ERT). Patient 5rejected his first graft which wasfollowed by an escalation ofantibody titers. This immuneresponse resolved after a sec-ond transplant on day 328.

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due to antibodies, we assessed the enzyme inhibition inthe same patient before and after HSCT i.e. the specimenwith the highest titer of antibodies and no detectable anti-bodies on ELISA. The data confirmed that there was nocatalytic inhibition in the absence of antibodies (Figure 2B).

Inhibition of cellular uptake of catalytically activeenzymeCellular uptake inhibition was also assessed in the sam-

ples with the highest antibody titer for each patient in thelongitudinal series. Inhibition of enzyme uptake by MPSIfibroblasts was seen in 75% (n=6) of patients in this series.Figure 3A illustrates the results of the uptake inhibitionassay in eight patients in the longitudinal series. Twopatients (patients 6 and 8) did not show any inhibition. Itis interesting to note that one of these patients (patient 6)had evidence of some enzyme inhibition on catalytic inhi-bition assays. In contrast the two patients (patients 4 and5) who did not have any evidence of catalytic inhibitionshowed 25% and 76% inhibition of enzyme uptake,respectively. Patients 2 and 7 who showed over 75% inhi-bition of enzyme activity also demonstrated high levels ofcellular uptake inhibition (over 70%). There was no corre-lation between the catalytic inhibition and cellular uptakeinhibition in the rest of the patients suggesting that theantibodies are polyclonal. The cellular uptake inhibitionassay was repeated in non-tolerized specimens (patient onERT pre-transplantation and with a high-titer immuneresponse) and tolerized specimens (post-HSCT with noantibodies) from the same patient to confirm that the inhi-bition was due to the antibodies in the serum (Figure 3B).

Immune response and disease biomarkersLongitudinal biomarker data (DS/CS ratio) were avail-

able for six patients in this series. Figure 4 shows the rela-tionship between antibody titers and DS/CS ratio over thefollow-up period. Patient 2 showed an immediateimprovement in DS/CS ratio after starting ERT. Thisimprovement in biomarkers was halted by a high-titerimmune response. This pattern continued even after theHSCT was performed and a further improvement inDS/CS ratio resumed only after the immune response wascompletely abrogated (Figure 4A). The DS/CS ratio inpatient 3 stayed high despite HSCT, with a continuinghigh-titer immune response. The slight improvement in

DS/CS ratio seen in this patient was suboptimal and couldbe considered as a therapeutic failure at this stage. TheDS/CS ratio after resolution of the immune response is notyet available for this patient (Figure 4B). The improvementin biomarker levels in patient 4 (Figure 4C) was significantafter ERT was commenced; however, this progress washalted and partially reversed after a high-titer immuneresponse was generated. The subsequent improvement inDS/CS ratio followed resolution of the immune response.Patient 5 rejected his first transplant and was given a sec-ond transplant which was succcessful. His ERT was inter-rupted during this period. The initial improvement in bio-marker levels plateaued during the high-titer immuneresponse despite restarting ERT. After the second HSCT,the biomarker levels showed some deterioration despiteERT and HSCT and improved only after the immuneresponse was completely resolved (Figure 4D). Patient 6showed a suboptimal biomarker response to ERT duringthe high-titer immune response (Figure 4E). The DS/CSratio in patient 7 dropped significantly after ERT but fur-ther follow-up data were not available during the immuneresponse and following the HSCT (Figure 4F).

Discussion

The immune response to replacement recombinanthuman proteins has been widely reported.4 However, theclinical impact of antibodies ranges from no significanteffect23 to almost complete failure of therapy.24 HemophiliaA and B are examples of congenital disorders in which theimmune response (inhibitory antibodies) can make the



Figure 2. (A) Catalyticenzyme inhibition in eightpatients (1-8) comparedto a standard (normalserum) across variousenzyme concentrations.(B) Inhibition due to toler-ized and non-tolerizedserum. Non-tolerizedserum taken from apatient (patient 3) on ERTbefore HSCT with high titerantibodies is compared totolerized serum takenfrom the same patient ayear after HSCT (with noantibodies) which showsno significant inhibition.

Table 3. Summary of immune responses in the longitudinal series ofpatients.

Median time to first positive ELISA test 38.5 days (range 14-129)Median time to highest immune response 55.5 days (range 17-129)Median time to immune tolerance 101 days (range 26-137)Incidence of high titer antibodies 87.5% (7/8)Incidence of catalytic inhibition 62.5 (5/8)Incidence of cellular uptake inhibition 75% (6/8)Immune tolerance 1 year post-HSCT 100% (28/28)

A B% Enzyme inhibition compared to standard Patient 3 (during treatment)

Patient 3 (tolerized)

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Figure 3. (A) Cellular uptakeinhibition in all patients (1-8)in the longitudinal series isshown. Patients 6 and 8showed no cellular uptakeinhibition. (B) Tolerizedserum (post-transplant withno antibodies) showed noinhibition compared to sig-nificant inhibition by non-tolerized serum (prior totransplant) in the samepatient.

Figure 4. Correlationbetween biomarkers (DS/CSratio) and the antibody titersin six patients in the longitu-dinal series (patients 2-7).The shaded areas representthe period of ERT and blackarrows point to the time ofHSCT. (A) Patient 2 showedan immediate improvementin the DS/CS ratio afterstarting ERT which was halt-ed by high titer anti-IDUAantibodies. The biomarkerlevels improved after theimmune response was abro-gated. (B) Patient 3 showedminimal improvement inbiomarkers despite HSCTand ERT during the high-titerimmune response. (C) Theimprovement in biomarkerlevels in patient 4 was signif-icant after ERT was com-menced; however, thisprogress was arrested andreversed after the high titerimmune response was gen-erated. The biomarkersimproved after resolution ofthe immune response. (D)Patient 5 rejected his firstHSCT and had a second suc-cessful transplant. The initialimprovement in biomarkerlevels plateaud during thehigh-titer immune responsedespite ERT (note a briefperiod when ERT was inter-rupted after the first failedHSCT) which improved onlyafter the immune responsewas completely resolvedafter the second HSCT. (E)Patient 6 showed a subopti-mal response to ERT duringthe high titer immuneresponse. (F) The DS/CSratio in patient 7 dropped toa significant level but furtherfollow-up data were notavailable for the period dur-ing the immune responseand following the HSCT.Biomarker data are notavailable for patients 1 and8.

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replacement therapy (recombinant human factor VIII andIX) virtually ineffective. It is noteworthy that the trueimpact of neutralizing antibodies can only be measuredaccurately using robust functional assays, such as theBethesda assays for hemophilia inhibitors. It is consider-ably more challenging to study the impact of antibodies inLSD because of the absence of standardized immuneassays and the marked phenotypic heterogeneity in thenatural course of a rare illness. While we studied only twomechanisms of enzyme neutralization, it is hypothesizedthat antibodies against ERT can inhibit the treatment byseveral mechanisms.25 The polyclonal nature of the IgGantibodies makes it imperative to develop multiple func-tional assays to encompass the full range of effects due toantibodies.26 Despite the heavy financial burden of ERT,there have been no collaborative efforts to develop andstandardize quantitative and functional assays. As a con-sequence the true incidence and impact of these allo-anti-bodies remain unknown. In recent years, the functionally active nature of allo-

antibodies and the catastrophic effects of the alloimmuneresponse in some LSD11-12 were recognized and led toefforts to develop an effective and quick immune toler-ance induction regimen. These strategies had variable out-comes and serious side effects were reported in somecases.13,27-29 There is a risk that intensifying the toleranceinduction protocols by combination chemotherapy andimmune suppression would result in significantlyincreased morbidity, as has been reported in hemophil-ia.30,31 Development of immune tolerance with allogeneicHSCT has been reported in various autoimmune disordersand to date over a thousand HSCT have been carried outfor this indication.32 The use of low intensity conditioningregimens, the ability to manipulate the graft and the verylow rate of transplant-related mortality have led to the useof allogeneic HSCT as a long-term tolerance inductionstrategy in some solid organ transplant recipients.33,34 Therole of HSCT as an immune tolerance induction mecha-nism in patients with neutralizing allo-antibodies has notyet been established. All the patients in our study had the severe phenotype

of MPSI H (Hurler’s syndrome). Seven patients werehomozygous for null mutations, conferring a high risk ofallo-immune responses. Consistent with previous reports,we found a high incidence of antibodies in this subgroup.All transplant recipients in the longitudinal series clearedtheir antibodies to clinically insignificant levels within amedian period of 101 days despite normal enzyme levels.None of the 20 patients in the cross-sectional group had ahigh-titer immune response 1 year or more after HSCTand cessation of immunosuppression. Full donor engraft-ment was not required for immune tolerance as somepatients were tolerized despite achieving only mixedchimerism. These data confirm that allogeneic HSCT is aneffective and quick mechanism of inducing immune toler-ance. These data also have important implications forCD34 gene therapy. In animal models of LSD and hemo-philia, stem cell transplantation after hematopoietic stemcell-based gene therapy tolerized the immune system tothe infused protein.35,36 The majority of patients (62.5%) demonstrated evi-

dence of some catalytic inhibition by antibodies. Eventhough the catalytic inhibition appears weak at highenzyme concentrations, this in vitro inhibition may reflect

partial neutralization of infused enzyme due to the veryshort half-life of Aldurazyme (3 h in the absence of anti-bodies) and a mean maximum plasma concentration (Cmax)of 1.2-1.7 mg/mL (ALID-014-02: A phase II Open-LabelClinical Trial of Aldurazyme). The inhibition of endoge-nous (human and mouse) enzyme by antibodies suggestscross-reactivity of anti-IDUA antibodies to endogenousIDUA. This signifies a need for HSCT-induced immunetolerance, as the presence of antibodies at high titers afterHSCT would potentially neutralize the cellular therapy(enzyme delivered by allogeneic HSCT).The cellular uptake inhibition can be demonstrated at a

much higher enzyme concentration. We used enzymeconcentrations of less than half the Km (100 ng/ml) tooptimize the uptake inhibition and serum at 1:100 dilu-tion. Corrected for dilution, this is equivalent to just oversix times the Cmax (maximum plasma concentration afterinfusion) of Aldurazyme, making the treatment virtuallyineffective in some patients with high-titer immuneresponses. The cellular uptake inhibition at a higherenzyme concentration compared to catalytic inhibitionsuggests stronger inhibition of the mannose-6-phosphatebinding sites (and enzyme uptake) than the catalytic siteof recombinant IDUA by anti-IDUA antibodies. Ourstudy looked at the antibody neutralization of enzyme ina specific group of MPSI patients with a greater propensityto develop a high-titer immune response. It is possible thatthe variable effects and polyclonal nature of anti-IDUAantibodies might have resulted in underestimation of theeffects of antibodies in previous studies because of thepooling of data from various groups of patients. It is,therefore, important to assess these patients individually. Analysis of biomarker data and antibody titers show

that the recovery is slowed or, in some cases, reversed inthe presence of a high-titer immune response. Generallythere appears to be a close relationship between the two,although the DS/CS ratio and biomarker responses usuallylagged behind antibody responses by several days, con-firming the in vivo neutralization of ERT by antibodies. In conclusion, our data show that the high-titer immune

responses in MPSI H patients treated with ERT can neu-tralize replacement therapy in a significant proportion ofpatients. In the past, the heterogeneous clinical course ofthe disease compounded by a lack of robust biomarkersand reliable functional immune assays made it very diffi-cult to evaluate the effect of antibodies in LSD patientstreated with ERT. There is now a dire need to standardizequantitative and qualitative immune assays in thesepatients. Given the remarkable improvement in the out-come of HSCT, this therapy is now a viable therapeuticmodality as a mechanism for inducing immune tolerancein patients with refractory immune responses to ERT andother replacement therapies by substituting the enzyme-naïve immune system with that of the donor. The gener-ation of high-titer neutralizing antibodies to ERT prior toHSCT makes it unnecessary to continue ERT infusions inthe presence of an immune response, particularly if thetransplant is carried out early after the diagnosis and in theabsence of any serious co-morbidities. However,inevitably, some clinically unstable patients will benefitfrom ERT to optimize their clinical status prior to HSCT.Close biochemical and clinical monitoring of the immuneresponse in ERT-treated LSD patients can help to deter-mine the optimum therapy for this group of patients.



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Authorship and Disclosures

The information provided by the authors about contributions frompersons listed as authors and in acknowledgments is available with

the full text of this paper at www.haematologica.org.Financial and other disclosures provided by the authors using theICMJE (www.icmje.org) Uniform Format for Disclosure ofCompeting Interests are also available at www.haematologica.org.

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2. Connock M, Juarez-Garcia A, Frew E,Mans A, Dretzke J, Fry-Smith A, et al. Asystematic review of the clinical effective-ness and cost-effectiveness of enzymereplacement therapies for Fabry's diseaseand mucopolysaccharidosis type 1. HealthTechnol Assess. 2006;10(20):iii-iv, ix-113.

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