Paul Imbach

Paul Imbach
Editor
Antibody Therapy
Substitution –
Immunomodulation –
Monoclonal Immunotherapy
123
Antibody Therapy
Paul Imbach
Editor
Antibody Therapy
Substitution – Immunomodulation –
Monoclonal Immunotherapy
Editor
Paul Imbach
Department of Pediatrics
Medical Faculty of the University of Basel
Basel
Switzerland
ISBN 978-3-319-68037-8 ISBN 978-3-319-68038-5 (eBook)

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The path of antibodies therapy from
substitution to immunomodulation to the
development/use of monoclonal antibodies
for patients with immune deficiencies,
inflammatory, autoimmune and oncological
diseases – based on the similarities of the
immune pathogenesis, namely the loss of
immune tolerance – is presented
Foreword
In 1735, Werlhof described a clinical syndrome of bleeding and purpura long before
platelets were identified as the cellular component of blood that play an essential
role in primary hemostasis. Werlhof’s disease, as it became known, was later
renamed idiopathic thrombocytopenic purpura, from which the acronym ITP origi-
nally derives. Little followed from these observations until the early 1900s, and we
have just passed the centenary of the first successful treatment for the condition. In
1916, a medical student in Prague, Paul Kaznelson, proposed that, in an analogy
with hemolytic anemia, essential thrombocytopenia, as it was also known, resulted
from increased platelet destruction in the spleen. Kaznelson convinced his tutor to
perform a splenectomy in a 36-year-old woman with a history consistent with our
current definition of chronic ITP. The platelet count was 2 × 109/l prior to splenec-
tomy and rose to 500 × 109/l within four weeks from surgery with complete resolu-
tion of the purpura. This confirmed the role of the spleen in the pathophysiology of
ITP, and splenectomy has remained a mainstay of treatment ever since. The patho-
physiology of ITP remained elusive for many decades. Although some intriguing
observations by Dameshek and Miller in 1946 suggested reduced megakaryocyte
function, the “increased platelet destruction, reduced production” debate appeared
to have been settled by the classic Harrington-Hollingsworth experiments in 1951
that unequivocally demonstrated that ITP was characterized by reduced platelet sur-
vival due to a humoral factor that was soon identified as an antiplatelet antibody. In
his historical review of ITP in 2002, Paul Imbach reported that Harrington et al. had
also observed a child with purpura born to a mother with chronic ITP that resolved
in the child 3 weeks after birth, although the mother still had ITP, indicating that a
humoral antiplatelet factor had been passed from mother to child.
At the same time, the successful use of corticosteroids and adrenocorticotropic
hormone (ACTH) in elevating the platelet count was described by Wintrobe (1951),
and standard-dose prednisolone has been considered the initial treatment for newly
diagnosed ITP since then. Immunosuppressive agents were introduced in the 1960s,
when the autoimmune nature of ITP was clarified.
A milestone in the treatment of symptomatic ITP in children, however, was the
introduction of intravenous immunoglobulin by Paul Imbach in 1981. The efficacy
of this treatment was subsequently validated both in adults and in pregnancy by
Adrian Newland in 1983. Abdulgabar Salama introduced anti-D treatment and the
vii
viii Foreword
concept of macrophage blockade in 1984. James Bussel and his group later expanded
the knowledge about the modalities of treatment with anti-D in various settings.
With an increasing understanding of the underlying molecular biology and with
advances in pharmacological technologies, targeted therapy became more attractive
and has been investigated since the 1980s in many conditions. In ITP, the most con-
sistent results with monoclonal antibody therapy have been obtained with ritux-
imab, an anti-CD20 chimeric antibody inducing B-cell depletion. Roberto Stasi first
reported the successful use of rituximab in adults with chronic ITP in 2001. This
agent has become the standard (albeit unlicensed) treatment for patients with this
condition in many countries, and its use has been extended to a variety of autoim-
mune conditions.
There is no doubt that in recent years, we have seen a major breakthrough in the
treatment of chronic ITP, with the introduction of the thrombopoietin receptor ago-
nists. The pioneering work of David Kuter with these agents has shown response
rates unequalled by previous medical therapies. These agents are almost as effica-
cious in splenectomized patients as in the non-splenectomized ones, and recent
studies have confirmed the efficacy and safety following long-term usage.
The second half of the twentieth century brought recognition on the autoimmune
components of ITP, hence the need for a new standard nomenclature, which has
been widely accepted. ITP currently stands for immune thrombocytopenia, a name
that more appropriately reflects the low platelet count rather than purpura as the
main feature of the disease and defines its underlying nature.
Advances in our knowledge of the disease have paralleled the burgeoning avail-
ability of new therapeutic agents, and we are now entering an era of treatment
options based on pathophysiological principles. There is no doubt that the enormous
expansion in our understanding of the condition and its treatment was stimulated by
the observations of Paul Imbach in children with thrombocytopenia. A relatively
rare disease with few treatment options, the disease suddenly became totem for
clinical study and laboratory investigation and a marker for the possibilities in other
autoimmune diseases. It was Imbach’s realization that intravenous immunoglobulin
was more than a replacement treatment but that it had a major impact on both immu-
nological and phagocytic functions that had implications in a wide variety of condi-
tions. This book systematically charts the history and the development of
immunoglobulin and its association with ITP while highlighting how treatment and
understanding of the latter has changed and how the former has developed into an
important therapeutic option. Our forebears would be astounded at the progress
over the last 50 years which is admirably described in these chapters.
Adrian Newland, CBE, MA, FRCP, FRCPath

Academic Haematology Unit
Barts and The London School
of Medicine and Dentistry
Queen Mary University of London
Turner Street, London, E1 2AD, UK
Contents
1 The Clinical Translation of Intravenous Immunoglobulin

from Substitution to Immunomodulation�� 1
Paul Imbach
Part I Update of Substitutive and Immunomodulatory

Antibodies/Drugs Indications
2 From Immune Substitution to Immuno-modulation�� 15
Volker Wahn
3 Manual of Primary and Secondary Immunodeficiencies�� 23
Paul Imbach and Volker Wahn
4 Manual of Intravenous and Subcutaneous IgG Indications
in Autoimmune Diseases�� 35
Paul Imbach
5 Anti-D: A Type of IVIg�� 61
Ramsha Khan and Alan H. Lazarus
6 Mechanisms of Action and Immunomodulation by IVIg�� 73
Alan H. Lazarus
7 Immunomodulatory Drugs and Monoclonal Antibodies�� 85
Howard A. Liebman
8 Use of Intravenous Immunoglobulin in Neurology�� 101
Marinos C. Dalakas
9 Use of Intravenous Immunoglobulin in Dermatology�� 111
Jochen H.O. Hoffmann and Alexander H. Enk
Part II Basics of IgG Concentrates

10 Historical Aspects of Polyclonal IgG Preparations�� 121
Volker Wahn and Peter Späth
ix
x Contents
11 Basics of Immunoglobulins as Effector Molecules and Drugs�� 133

Tchavdar L. Vassilev, Victor Kostov, Stephan von Gunten, and
Anastas D. Pashov
12 Essentials of the Production of Safe and Efficacious
State-of-the-Art Polyclonal IgG Concentrates�� 151
Peter J. Späth
13 Current IgG Products and Future Perspectives�� 175
Peter J. Späth
Part III Immune Thrombocytopenia: The First

Immunomodulatory IgG Treatment
14 Updates in Immune Thrombocytopenia: Terminology,
Immunomodulation and Platelet Stimulation,
and Clinical Guidelines and Management�� 205
Cindy Neunert
15 Health-Related Quality of Life in Patients with Immune
Thrombocytopenia �� 213
Robert J. Klaassen and Nancy L. Young
16 ITP in Childhood: Predictors of Disease Duration �� 223
Carolyn M. Bennett
17 Immune Functions of Platelets �� 241
Rick Kapur and John W. Semple
18 Thrombopoietin Receptor Agonists: Characteristics,
Adverse Effects, and Indications�� 261
Jenny Despotovic and Amanda Grimes
19 Registries in Immune Thrombocytopenia: The History
of the Intercontinental Cooperative ITP Study Group�� 277
Thomas Kühne
Part IV Translation from Polyclonal to Monoclonal Antibody Treatment

20 Therapeutic Monoclonal Antibodies as Immunomodulators and Anti-
Cancer Agents: Development, Evidence of Efficacy, Mechanisms
of Actions, Adverse Effects�� 291
Tim Niehues
21 Monoclonal Anti-CD20 (B-Cell) Antibody and
Autoimmune Diseases�� 343
Bertrand Godeau
About the Editor
Paul Imbach is a highly respected pediatric oncologist-hematologist who has

developed worldwide clinical research on the indications for intravenous immuno-
globulin. Dr. Imbach graduated in Medicine in 1972. He went on to structure the
Swiss Pediatric Oncology Group (SPOG) and in 1978 performed the first autolo-
gous stem cell transplantation in Switzerland. He discovered the immunomodula-
tory effects of human immunoglobulin G concentrate (IVIG) in childhood immune
thrombocytopenia (as reported in Lancet in 1981). In 1990, Dr. Imbach began work-
ing at the University Children’s Hospital Basel, where he started stem cell trans-
plantation in children and was appointed Head of Pediatric Oncology-Hematology.
He was subsequently elected as full professor and also as Dean of Education intro-
ducing a thorough curriculum reform at the medical faculty of the University of
Basel. In parallel to his other activities, he co-founded the International Cooperative
ITP Study (ICIS) group (www.itpbasel.ch), which now has more than 90 centers
worldwide. He has served as president of medical societies and foundations, is a
member of medical editorial boards, and has published over 350 peer-reviewed
articles, textbook chapters as well as the textbook Pediatric Oncology – A
Comprehensive Guide (3rd edition 2014) in German and English. In 2015 he was
awarded the Guido Fanconi Prize, the highest award of the Swiss Society of
Pediatrics.
xi
Introduction
The book starts with a narrative description including citations of the first clinical
observation of immunoglobulin (IgG) administration in children with “idiopathic”
thrombocytopenia. This highlights the importance of clinical observation, inquisi-
tiveness and translational clinical research. This leads into a discussion of the fun-
damental discovery (Chap. 2).
Written in a practical fashion as manual, Chap. 3 describes some main indica-
tions of substitution by IgG in primary and secondary immune deficiencies and
Chap. 4 summarizes many of the new immunomodulatory indications, some of
which remain quite controversial.
Autoimmune disorders are characterized by complex heterogeneity of clinical
presentation and pathophysiological abnormalities of the innate and adaptive
immune system. Immunomodulatory IgG indications are rarely evidence based and
in general are disorder oriented with specific individual indications. Based on the
very large number of clinical and laboratory studies in the literature—over 40,000
peer-reviewed articles in Pubmed—a categorization of the indications is proposed
in the manual of autoimmune disorders.
One specific IgG preparation is Anti-D, which is a targeted product specifically
binding to the Fc receptors as its mechanism of action; in contrast the polyclonal
IgG concentrate induces a broad spectrum of synergistic immune challenges to the
imbalanced immune system in patients with autoimmune disorders.
Chapter 6 is dedicated to the general immunomodulatory effects of IgG followed
by a chapter that covers classical drugs, IgG and monoclonal antibodies with explo-
ration of their mechanisms of action. The combination of the different immuno-
modulators often results in a more effective clinical outcome in the individual
patient. The first part concludes with two expert reviews of the current use of IgG in
conjunction with other therapeutic options in both neurology and dermatology.
The second part of the book updates the basic knowledge of the IgG molecule
starting with historical aspects of polyclonal IgG. Currently production and the regu-
lations for a safe and effective IgG product are complex. Many such preparations are
now available internationally, and these are listed highlighting their specific charac-
teristics with a consideration of the future perspectives of IgG preparations.
Since ‘idiopathic’, now immune thrombocytopenia ITP was the key disorder of
the first observation of immunomodulatory effects of IgG, the third part summarizes
ITP as the model syndrome of autoimmune disorders. In the majority of children
xiii
xiv Introduction
with ITP the condition will resolve within weeks, months or very occasionally
years. In adults the position is more complex with few spontaneously remitting and
many developing chronicity. There has therefore been much interest in identifying
prognostic factors, studying clinical outcomes and reviewing health-related quality
of life issues in mild, moderate or severe disease. In order to standardize treatment
approaches guidelines have been developed and regularly updated. There is increas-
ing interest in secondary ITP and how it relates to the primary condition.
Newer aspects of platelet function are being recognized. Before 1980 the platelet
was mainly thought to be responsible for coagulation, but now it is increasingly
recognized as having an active role within the immune system (Chap. 17).
For many years the role of megakaryocytes has been suspected in the pathology
of ITP, and the recognition of reduced platelet production led to the development of
platelet stimulation by recombinant thrombopoietin and thrombopoietin receptor
agonists, which is the focus of Chap. 18. For patients with severe, chronic ITP, e.g.
with recurrent or at risk of life-threatening bleeding, thrombopoietin receptor ago-
nists have become a major option with a low adverse event profile and increasingly
have a place early in the treatment of refractory or relapsed disease. Chapter 18
summarizes the development and the characteristic of this long-term approach.
Nevertheless, in patients with acute, life-threatening bleeding immediate high
dose IgG and/or corticosteroid administration and occasionally platelet transfusion
remain the first choice.
The heterogeneity and immunological complexity of autoimmune diseases was the
reason to start worldwide online registries of patients with ITP. The first endpoint of
these registries is to distinguish subgroup of patients concerning demographics and
follow up of this rare disease (for details see Chap. 19 and www.itpbasel.ch). There is
also a large adult registry in the UK (www.ukitpregistry.com). Through recognition of
subgroups of an autoimmune disease, evidence-based trials might become feasible.
We are now entering an exciting new phase of a “bridge” from antibody therapy of
human origin progressing to monoclonal, engineered (or human adapted, e.g. CAR
cell) treatment as an immunomodulatory approach to both autoimmune disorders and
cancer. In a critical overview Chap. 20 explains the definitions, methods and adverse
effects of monoclonal antibodies and presents an extensive list of those currently
available monoclonal antibodies and their possible indications. One of the first anti-
bodies introduced into clinical use, anti-CD-20, is described in Chap. 21. The anti-CD
20 antibody was initially developed as an adjunct in the treatment of Non-Hodgkin
Lymphoma NHL, but its activity against immune competent B lymphocytes led to its
exploration in many immunological and oncological disorders—based on the simi-
larities of the immune pathogenesis, namely the loss of immune tolerance.
In summary the use of IgG, monoclonal antibodies and a variety of combinations
with other immunomodulatory approaches has opened up the path from translation
to more targeted biological, therapeutic approaches for patients with unresolved
immune and malignant disease.
We thank all our contributing authors and the staff of Springer, especially Mrs. Meike
Stoeck and Mrs. Dr. Isabelle Arnold, for their commitment to this extraordinary book.
Basel, Switzerland Paul Imbach

The Clinical Translation of Intravenous
Immunoglobulin from Substitution 1
to Immunomodulation
Paul Imbach
The subject of the book highlights 37 years of experiences following the first observa-
tion emphasizing the importance of the skill of critical medical observation, the devel-
opment and production of a safe human blood extracts with minimal adverse effects,
and research on how the administration of IgG intravenously or subcutaneously bene-
fits patients with other autoimmune disorders and the potential mechanisms of action.
1.1 History of New Observations
1980 Pediatric Hematology-Oncology, University Children’s Hospital Berne,

Switzerland: On January, during a ward visit PI (the abbreviation of names relates to
the full names in the reference list) and his colleagues observed a minimal increase in
platelet count after each intravenous immunoglobulin G (IVIG) substitution in a child
with typical Wiskott-Aldrich syndrome who also had thrombocytopenia in addition to
hypogammaglobulinemia. On the same ward, there was a 12-year-old boy (M) with
severe, refractory immune thrombocytopenia ITP of 9 years’ disease duration with
many complications despite splenectomy and cytotoxic treatment. The latter treat-
ments resulted in secondary hypogammaglobulinemia and recurrent infections.
Discussion with visiting colleagues led to the decision to ask for permission from the
medical director ER to administer IVIG, and for consent from the patient with ITP and
his parents. The first dose of 0.4 g IVIG/kg body weight was administered on the next
Monday. During the evening visit, the boy said: “I am feeling much better, and I am
sure that my platelet count will be much better tomorrow.” This was the reality: his
platelet count increased remarkably from 2 to 21 × 109/L. Now, PI discussed the
observation with the chief immunologist SB treating adults with primary immunode-
P. Imbach
Medical Faculty of the University of Basel, Basel, Switzerland
e-mail: [email protected]
© Springer International Publishing AG, part of Springer Nature 2018 1

P. Imbach (ed.), Antibody Therapy, https://doi.org/10.1007/978-3-319-68038-5_1
2 P. Imbach
ficiency with IVIG at the cancer research institute, who recommended to empirically
continue the daily administration of the same dose of IVIG. On Friday of that week
and after 5 × 0.4 g IVIG/kg body weight, the boy’s platelet count was higher than
150 × 109/L.
In the following PI asked for permission to administer the same IVIG treatment
regimen to other patients with severe, chronic ITP with platelet counts of less than
30 × 109/L and without hypogammaglobulinemia and as a control for two children
with aplastic anemia. The IVIG was provided by the Swiss Red Cross, who were the
local producers of the product. At this time, gamma globulin was the former name of
IVIG.
In this pilotstudy children with ITP consecutively responded to IVIG, but chil-
dren with aplastic anemia did not. Following these observations, the first manuscript
was produced with input of his consultant HpW (Imbach et al.; see some citations
(in cursive letters) and the Fig. 1.1 from the very first article below):
Summary
A new immunoglobulin (IgG) for intravenous use was given in high doses to 4 children
with refractory idiopathic thrombocytopenic purpura (ITP) and 2 children with idiopathic
aplastic anemia (IAA). Within 5-10 days after initiation of IgG therapy the platelets of the
children with ITP rose to 300,000-650,000/mm3 and could be maintained at normal levels
with one IgG infusion every 1-3 weeks. No response of platelet counts, was observed in the
2 patients with IAA… The IgG treatment was tolerated without complication by all patients.
The effect of intravenous IgG on the number of platelets is shown in Fig. 1. (see
below). The platelets of all patients with ITP rose to a maximum between 300,000 and
650,000 within 5 to 10 days and returned to values between 100.000 and 300,000/mm
3 within the next 10 days.
The platelet count of patients with aplastic anemia was not influenced by the IgG ther-
apy during the period of observation.
Discussion
We do not know how i.v. IgG administration influences the elimination of platelets. One
could postulate that IgG acts primarily on the reticuloendothelial system and diminishes its
platelet-eliminating effect. This hypothesis would also explain why the non-splenectomized
child with chronic ITP (patient 3) required more frequent IgG infusions than the two sple-
nectomized patients with chronic ITP in order to maintain adequate platelet levels. We also
do not know whether infused lgG has a different effect on platelets of children with chronic
versus acute ITP. It should be noted that the child with acute ITP resistant to prednisone,
after a single 5-day course of IgG, remained in unmaintained remission for at least 6 weeks.
Despite the fact that IgG had no effect on the platelet counts of our 2 patients with
aplastic anemia, further trials, particularly in patients with immune aplastic anemia may
be rewarding.
Finally, since the intravenous IgG therapy described was well tolerated and had a strik-
ing effect on the platelet count of 4 children with chronic or acute ITP, the question arises
whether the use of intravenous IgG should be considered for other autoimmune diseases as
well. Obviously, the mechanism by which intravenous IgG exerts its effects should be known
more precisely (end of citation).
In parallel with the first publication, the investigators continued to treat a total of
13 children with acute (7 patients) and chronic (6 patients) ITP using the same treat-
ment regimen. Because all children in this pilot study showed responses to IVIG,
the statistician determined the consecutive response rate to be a new phenomenon.
1 The Clinical Tranlslation of IVIG from Substitution to Immunomodulation 3
Fig. 1.1 Effect of i.v. IgG: Patients 1–4 with refractory ITP: 0, 4 g i.v. IgG/kg body weight/day x 5
rose to 300–650 x 103/mm3 platelet counts within 5–10 days and could be maintained at normal
levels with one dose of i.v. IgG every1–3 weeks. Patients 5 and 6 with idiopathic aplastic anemia:
no reaction of platelet counts to the same doses of i.v. IgG
4 P. Imbach
The group sent their manuscript to The Lancet. The editor in chief of The Lancet,
made confirmation of the originality of the new observation, published it as a rapid
communication. In the article and Fig. 1.2a and b (Imbach et al. 1981), it was stated
(citations slightly modified):
‘Patients and Methods’
All patients first received, on 5 consecutive days, 0.4g IVIG/kg body-weight/day. IVIG is
a polyvalent Ig concentrate obtained by modified alcohol cryoprecipitation, including mild
acidification at pH4. The similarity of its in vivo biological half-life with that of normal
serum IgG, and its intact Fc-receptor mechanisms reflect the structural and functional
integrity of the 7S-IgG.
‘Results’
No patient had adverse effects during and/or after immunotherapy.
IVIG induced a dramatic initial response in all patients (Figs. 1 and 2). In twelve of the
thirteen children, the platelet count rose from pretreatment counts of <30x109/l platelets to
a maximum of 150–600 x 109/l within 5-10 days of onset of treatment and returned to
80-400/ during the next ten days. In the thirteenth patient (patient 7), maximum counts were
achieved after 10 days. Serum IgG levels rose to >2000mg/dl 10-20 days after onset of
IVIG treatment (Figs. 1 and 2).
‘Discussion’
The dramatic response to IVIG in patient 1 prompted us to give IVIG to other patients
with chronic ITP and, later, to patients with acute ITP.
Although all of our patients showed a dramatic initial response to IVIG, the rates of
increase and decrease and the maximum platelet counts differed between patients.
In view of the large IgG doses given, the mode of action of IVIG could be the over-
loading and blocking of the reticuloendothelial system by IgG catabolism. This expla-
nation might account for the differences in the response patterns between
splenectomized and non-splenectomized children with chronic ITP. Reaction with and
inactivation of circulating antiplatelet factor or interference with platelet-bound IgG
and/or C-3, could be responsible for immediate effects, and activation of T and sup-
pression of B cells for late effects of IVIG. In one patient with acute ITP (not included
in this study) 0.5 g of a pepsin-treated gammaglobulin (Fab’)2/kg body-weight/day on
3 consecutive days did not influence the platelet count, whereas a single dose of 0,4 g
of IVIG/kg body-weight raised counts from 1.7 to 6x109/l within 6 h and to 12.6x109/l
within 18 h.
How IVIG works still needs to be investigated. The most effective and economic dose
will also have to be determined.
This article has been followed up by one for adults with ITP at the neighbor-
ing university (Fehr et al. 1982), by two other hemato-immunologists (Newland
et al. 1983; Abe et al. 1983), and another colleague (Bussel and Hilgartner
1984). All confirmed the effects reported in the first publications. The observa-
tions led to much speculation on the many potential mechanisms of action and
stimulated worldwide interest and study from clinical and laboratory investigators
(see part III).
The nonprofit producer of IVIG was met with a high demand for the product and
proposals for IVIG studies. Sandoz (later named Novartis), as a professional, world-
wide distributor and coordinator of IVIG, took over that demand, while the local
Red Cross expanded human-derived IVIG production, development, and research.
The name changed from intravenous IgG to Sandoglobulin.
Fig. 1.2 (a) Patients with chronic or intermittent ITP (b) Patient with acute ITP
1981–1985 During a consultation at the University Children’s Hospital Basel,

the author met a well-known European expert of pediatric hematology EK, in
Ulm, Germany, to whom he presented his data described above. This hematolo-
gist showed a great interest in the new, therapeutic possibility of IVIG. At that
meeting, a proposal of an international cooperative study for administering IVIG
to children with acute, newly diagnosed ITP was agreed (see below). This study
was analyzed by BM and later published in “The Lancet” (Imbach et al. 1985).
The article is entitled “An international cooperative, randomized study compar-
ing IVIG with the classic corticosteroid treatment.” Here are some citations:
6 P. Imbach
Summary
In a randomized, multicentre study treatment with intravenous IgG was compared to
oral corticosteroids in 108 children with untreated acute immune thrombocytopenic pur-
pura. IVIG was an efficient treatment with no severe adverse reactions reported. The effects
of corticosteroids and IgG were identical for rapid responders, who accounted for 62% of
all patients. In contrast, patients requiring more than initial treatment responded better if
randomized to IgG. The serum levels increased two-fold after IgG. A significant rise in IgM
levels was observed after both IgG and corticosteroids.
Introduction
In a pilot study, the same preparation at a comparable dose was found to have a similar
effect in children with acute or chronic ITP and normal serum immunoglobulin levels. A
randomized trial was set up to compare the efficacy in raising platelet count, potential side-
effects, and the relapse rate and number of patients progressing to chronic ITP in previously
untreated children with ITP given intravenous IgG or oral corticosteroids.
Patients and Methods
After informed consent had been obtained from the parents, the patients were random-
ized according to a computer-generated code to receive either IgG 0.4 g/kg body weight
intravenously on 5 consecutive days or oral prednisone 60 mg/m2 daily for 21 days (initial
treatment). If the platelet count did not rise within the first 7 days (non-responder) or fell
below 30x109/l during the following 14 days (relapse), the patient was switched to the other
treatment regimen.
Results
47 children randomized to IgG and 47 to corticosteroids could be evaluated. The two
groups were well matched (see table below and Fig. 1.3)
CHARACTERISTICS OF TWO STUDY GROUPS
Corticosteroids IgG
n 47 47
M/F 22/25 23/24
Mean age 6 yr 3 mo 6 yr 10 mo
Mean initial platelat count (´109/1) (range) 9.8 (0.1–28) 9.3 (0.2–28)
Mean time from first symptom to
therapy (days) 16.8 13.0
History*
Postinfectious 33 38
Insidious 14 9
*No significant difference (p=0.337)
36 of 47 (77%) patients randomized to corticosteroids and 39 of 47 (83%) randomized

to IgG responded to the initial treatment (i.e., the platelet count rose to >100x109/l). The
mean time to the peak count was 12 days with corticosteroids and 9 days with IgG.
Of the 47 patients randomized to corticosteroids, 27 needed initial treatment only. 12 of
the 20 who required more than initial treatment crossed over to IgG, and in 5 patients the
platelet count rose to >100x109/l. Of the 47 patients randomized to IgG, 31 received initial
treatment only. 16 of 16 patients who required more than initial treatment were crossed over
to corticosteroids and 6 responded.
100
90
80
70
60
% 50
40
> 30 x 109/l platelets
30
IgG
20 Corticosterold
10
0.18 0.005 0.05 0.23 0.2 p-value
0
20 60 120 180 240 300 360
100
90
80
70
60
% 50
40
30
IgG
20 Corticosterold
10
0.09 0.03 0.20 p-value
0
20 60 120 180 240 300 360
100
90
80
70
60
% 50
40
30
IgG
20 Corticosterold
10
0.06 0.14 0.29 0.39 0.45 p-value
0
20 60 120 180 240 300 360
Days
Fig. 1.3 Percentage of patients with platelet count >30, >100, and >150 × 109/1
8 P. Imbach
The percentage of patients with a platelet count of >30x109/l, >100x109/l, or >150x109/l

at various times after starting therapy is shown for both treatment arms in Fig. 1. Significant
differences were found at days 60 and 120.
The serum IgG concentration increased by a factor of two from an average pretreatment
level of 12.5+/- 0.6 g/l to an average peak level of 25.9+/-0.9 g/l after five doses of IgG (fig. 3).
Peak values were observed between days 4 and 7. During the next 4 weeks the serum IgG
gradually returned to pretreatment levels.
In patients randomized to corticosteroids, the serum IgG concentration fell significantly
over 5 weeks from average levels of 12.0+/-3.7 g/l to 7.8+/-2.8 g/l- After initiation of ther-
apy, the difference in serum IgG concentration between the IgG and corticosteroid groups
was significant.
The serum IgM level increased significantly in both groups, but the rise was greater
(33%) in patients randomized to IgG (Fig. 1.4). 35 days after initiation of therapy the IgM
concentration had returned to pretreatment values.
Adverse reactions were observed during or shortly after 14 of 474 (2.9%) IgG infusions;
they consisted of headache (8 infusions) and/or fever (6), vomiting (3), and vertigo (3).
These reactions were observed in 14 of 63 (22%) of the children treated with IgG. In 47 of
61 (77%) children who received corticosteroids a Cushing’s syndrome developed initially
or later with an increase in body weight of more than 10% (28 patients), acne (3), and other
side effects (3).
Discussion
The best treatment for acute childhood ITP remains to be defined. The main aim is to
prevent potentially fatal central nervous system haemorrhage, which occurs in less than 1%
of all children with ITP admitted to hospital. 1 of the 108 children entered into our study
died from CNS haemorrhage, despite receiving three doses of IgG. Thus, if there is no rise
in platelets after one or two doses of IgG, the treatment does not prevent intracranial hem-
orrhage. At necropsy, this child had evidence of active disease.
In the prospective, randomized, double-blind, multicenter study Sartorius found that
corticosteroids, compared with placebo, accelerated the initial rise of platelet count but did
not significantly influence the further evolution of the disease.
Our results show that the intravenous administration of large quantities of structurally
and functionally intact IgG is an efficient treatment for acute ITP, including a rapid rise in
the platelet count in the majority of patients. Chronic ITP, defined as thrombocytopenia
(platelet count < 150x109/l) for more than 6 months, developed in 43% of patients random-
ized to corticosteroids and 32% of those randomized to IgG. If the platelet count defining
chronic ITP is reduced to <30x109/l, only 9 (19%) versus 4 (9%) patients met the criteria.
Indeed, this latter group of patients with platelet counts below 30x109/l required treatment
for longer.
The mechanisms by which corticosteroids and IgG act are unclear. Blockade of the
reticuloendothelial system (e.g., by modulation of Fc receptor expression or by Fc receptor
blockade), protection of platelet surface structure by monomeric IgG, or interference with
free or platelet bound antigen and/or immune complexes have been postulated.
There was no correlation between platelet-associated IgG index and the platelet count
of the serum IgG or IgM concentrations.
Despite IVIG treatment leading to significantly fewer side-effects and inducing a faster
response in slow responders than corticosteroids for ITP patients, IgG has not yet become
a generally accepted treatment for ITP, mainly because of the high costs.
In a preliminary study, we found that two infusions of 0.4 g IgG/kg body weight for rapid
responders achieved a satisfactory response and Bussel et al have shown that hospital
admission could be prevented by a single infusion of 1g IgG/kg body weight in about half
of children with acute ITP.
In the above-cited randomized study, it was not clear why IgM increased
after both IVIG and corticosteroid treatment. Additionally, the results on
IgG g/l
30
20
10
0.000 0.001 0.008 0.222

p-value
0.483 0.812 0.026 0.000
0
0 4–7 8–21 22–35 >35 days
n= 30 20 13 16 25
n= 29 21 9 17 24
IgM g/l
3
0.028 0.023 0.067 0.150

p-value
0.456 0.151 0.050 0.619
1
0 4–7 8–21 22–35 >35 days
n= 30 20 13 16 25
n= 29 21 9 17 24
Fig. 1.4 Serum IgG and IgM before, during, and after IgG (●) and corticosteroid (○) therapy.
p-values indicate significance of differences between initial values and values at various times after
initiation treatment
platelet-associated IgG (PAIgG, not cited above: see original article) are ques-
tionable; the sensitivity of the PAIgG test is high, but the specificity is low.
Therefore, the author began to collect serum samples from children with ITP,
and supplemented these with samples from adults provided by a colleague AN
in the UK. With these samples, the author traveled to the Scripps Institute in La
10 P. Imbach
Jolla, CA, where he could analyze PAIgG under supervision of RMcM (Imbach
et al. 1991).
As mentioned above, after the pilot study was published in The Lancet in 1981,
and after the start of the randomized study (Imbach et al. 1985), it was evident that
“the high-dose IVIG treatment” had similar effects as 2 × 0.4 or 1 × 0.8 g IVIG/kg
body weight in patients with ITP, doubling their serum IgG levels. A large, 4-arm,
randomized, cooperative study was organized by Canadian colleagues, comparing
0.8 g IVIG/kg and 2 × 1 g IVIG/kg body weight with a higher dose (4 mg/kg body
weight/day of corticosteroids during a short duration (4 days, then tapering) and
anti-D IgG treatment, which confirmed the lower dose of IVIG treatment in ITP
(Blanchette et al. 1994).
1986 After completing the analysis of the randomized study in children cited above
and the UK study in adults, the FDA in the USA and, later, the EMA in Europe
accepted IVIG treatment as a new therapeutic for ITP. Thus, ITP became the first
immunomodulary indication of IVIG as an autoimmune disorder.
1990 PI transferred to the University Children’s Hospital, Basel, where he contin-

ued his innonative work as head of pediatric oncology, hematology, and stem cell
transplantation, earned the title of full professor and as dean of education performed
a throughout curriculum reform at the medical faculty of the University of Basel.
1997 PI together with his colleague TK (Chap. 19) started the ongoing Intercontinental
Cooperative ITP Study (ICIS) group which now has over 90 cooperating centers
worldwide (www.itpbasel.ch).
1.2 he Translation of IVIG From ITP to Other Autoimmune

T
Disorders
Since the immunopathophysiology of ITP is similar in many other autoimmune and

chronic inflammatory disorders, IVIG became the subject of worldwide clinical and
laboratory studies and of its mechanisms of action. PI as an independent consultant
started to supported the clinical research and development group of the central labo-
ratory of the Swiss Red Cross and Novartis. Many peer reviewed articles, symposia
and presentations at congresses reflect the pros and contras the efficacies of IVIG
which the present book critically update.
1.3 The Bridge of Polyclonal and Monoclonal Antibodies
Furthermore the bridge to the engineered, monoclonal antibodies is in the focus of

this book by a basic knowledge, a updated list of target antibodies, their indication
and adverse effects. PI would like that the monoclonal antibodies would be com-
bined with the polyclonal IVIG in clinical trials with the endpoints of lower the side
effects and probably the increase of the efficacy. The high demand, the shortage of
IVIG and the high costs of polyclonal and monoclonal antibodies might be the
hindrance of such studies of antibodies combination.
Acknowledgment The author thanks Adrian C. Newland, London, UK, for his corrections and
suggestions of the chapter text.
References
Abe T, Matsuda J, Kawasugi K, Yoshimura Y, Kinoshita T, Kazama M. Clinical effect of intravenous
immunoglobulin on chronic idiopathic thrombocytopenic purpura. Blut. 1983;47(2):69–75.
Blanchette V, Imbach P, Andrew M, Adams M, McMillan J, Wang E, Milner R, Ali K, Barnard D,
Bernstein M, Chan KW, Esseltine D, de Veber B, Israels S, Kobrinsky N, Luke B. Randomised
trial of intravenous immunoglobulin G, intravenous anti-D, and oral prednisone in childhood
acute immune thrombocytopenic purpura. Lancet. 1994;344:703–7.
Bussel JB, Hilgartner MW. The use and mechanism of action of intravenous immunoglobulin in
the treatment of immune haematologic disease. Br J Haematol. 1984;56(1):1–7.
Fehr J, Hofmann V, Kappeler U. Transient reversal of thrombocytopenia in idiopathic thrombocy-
topenic purpura by high-dose intravenous gamma globulin. N Engl J Med. 1982;306:1254–8.
Imbach P, Barandun S, Baumgartner C, Hirt A, Hofer F, Wagner HP. High-dose intravenous gam-
maglobulin therapy of refractory, in particular idiopathic thrombocytopenia in childhood. Helv
Paediatr Acta. 1981a;46:81–6.
Imbach P, Barandun S, d’Apuzzo V, Baumgartner C, Hirt A, Morell A, Rossi E, Schoeni M, Vest
M, Wagner HP. High-dose intravenous gammaglobulin for idiopathic thrombocytopenic pur-
pura in childhood. Lancet. 1981b;317:1228–31.
Imbach P, Wagner HP, Berchtold W, Gaedicke G, Hirt A, Joller P, Mueller-Eckhardt C, Müller B,
Rossi E, Barandun S. Intravenous immunoglobulin versus oral corticosteroids in acute immune
thrombocytopenic purpura in childhood. Lancet. 1985;2(8453):464–8.
Imbach P, Tani P, Berchtold W, Blanchette V, Burek-Kozlowska A, Gerber H, Jacobs P, NewIand A,
Turner C, McMillan R. Different forms of chronic childhood immune thrombocytopenic purpura
defined by antiplatelet autoantibodies. J Pediatr. 1991;118:535–9.
Newland AC, Treleaven JG, Minchinton RM, Waters AH. High-dose intravenous IgG in adults
with autoimmune thrombocytopenia. Lancet. 1983;1(8316):84–7.
Further Reading
Imbach P, Lazarus AH, Kühne T. Intravenous immunoglobulins induce potentially synergistic
immunomodulations in autoimmune disorders. Vox Sang. 2010;98(3 Pt 2):385–94. Review.
Imbach P, Crowther M. Thrombopoietin-Receptor Agonists for Primary Immune
Thrombocytopenia. N Engl J Med. 2011;365:734–41.
Imbach P, Kabus K, Toenz O. Successful treatment of a severe drowning accident after 20 minutes
submersion. Schweiz Med Wochenschr. 1975;105(48):1605–11.
Imbach P, Odavic R, Bleher EA, Bucher U, Deubelbeiss KA, Wagner HP. Autologous bone mar-
row reimplantation in children with advanced tumor. First experiences of feasibility. Schweiz
Med Wochenschr. 1979;109(8):283–7.
Imbach P, Kuehne T, Arceci RJ. Pediatric Oncology: A Comprehensive Guide, Third Edition,
Springer 2014.
Part I
Update of Substitutive and
Immunomodulatory Antibodies/
Drugs Indications
From Immune Substitution
to Immuno-modulation 2
Volker Wahn
2.1 History
The description of agammaglobulinemia by Bruton (1952) was a milestone in the

history of medicine. For the first time it was shown that the absence of immuno-
globulins was associated with recurrent mainly bacterial and viral infections and
that the administration of Cohn fraction II subcutaneously (!) had the potential to
reduce the number and severity of such infections. Today, the treatment of severe
humoral immunodeficiencies consists in lifelong immunoglobulin replacement.
Intramuscular administration has become obsolete because of injection site-related
side effects but especially because only insufficient amounts of immunoglobulin
can be administered.
Intravenous administration of Cohn fraction II probably as a consequence of
complement activation by IgG aggregates offered no perspective. Thus, methods
had to be developed to make products well tolerated by patients. After appropriate
achievements, intravenous IgG (IVIG) replacement became the most widely used
route of administration since the 1980s and allowed the administration of adequate
immunoglobulin doses. IVIG treatment may be limited due to risk of anaphylactoid
reactions and poor vein access in small children and because health-care personnel
must, at least in Germany, directly supervise the infusions. As an alternative for iv
IgG replacement, rapid administration (up to 50 mL/h) pumps for subcutaneous
infusion (SCIG) are now generally accepted. SCIG was successively used for clini-
cal trials in the Scandinavian countries since the early 1990s. Both intravenous and
the subcutaneous administration achieve therapeutic IgG levels, and the clinical
efficacy is comparable. One SCIG uses local administration of human recombinant
hyaluronidase prior to IgG in order to increase the amount of IgG infused
V. Wahn
Department of Pediatric Pneumology and Immunology, Charité University Hospital,
Augustenburger Platz 1, 13353 Berlin, Germany

16 V. Wahn
subcutaneously and to reduce infusion numbers. All preparations are now approved
by many authorities, and the mode of treatment can be chosen on the basis of the
patients preferences.
2.2 IgG for Replacement: How Much?
Maintenance doses for IgG of between 400–1000 mg/kg body weight per month are
recommended. The major goal of treatment is the reduction of severe infections like
chronic rhinosinusitis or pneumonia and the avoidance of irreversible organ damage
like bronchiectases. The doses of IgG and the trough levels achieved vary among
patients depending on the underlying disease, the response to treatment, and the
presence or absence of chronic lung disease (Lucas et al. 2010). Probably, an indi-
vidualized approach comes closest to the patient’s needs.
Some general principles of treatment may reflect our current knowledge:
• There is a clear pharmacokinetic difference between iv and sc administration.

The trough level in iv therapy persists only a few days before the next peak level
is reached. In sc therapy peak and trough level hardly differ from each other.
From a recent meta-analysis (Orange et al. 2010), it was shown that the incidence
of pneumonia with IVIG declines up to a trough level of 1000 mg/dl. Similar
results were obtained with SCIG. A recent analysis from Holland (Janssen et al.
2017) shows that, at least in patients with CVID, trough levels >1000 mg/dl may
be required in order to prevent silent progression of airway disease. The authors,
however, state that randomized prospective trials would be necessary to prove
that trough levels >1000 mg/dl are, in fact, more effective. This illustrates that
current recommendations may have to be updated in the future.
• For now, if a patient with an IgG of 500–800 mg/dl is free from infections, the
maintenance of these trough levels is sufficient. If a patient with 1000 mg/dl still
suffers from severe infections, this trough level is not sufficient, and a higher
dose should be chosen. Thus, the optimum is reached with a biological and indi-
vidual IgG level that provides effective infection control (Bonagura et al. 2008).
• Most IgG licensing studies were done with XLA (X-linked agammaglobulin-
emia) and CVID (common variable immunodeficiency) patients together. Lucas
et al. (2010) have shown that an XLA patient needs significantly higher IgG
levels to be free from infections compared to patients with CVID. Thus, the
underlying disease must be taken into account.
• In patients with bronchiectases, all experts in the world agree that they require higher
doses of IgG than patients without this complication. Retrospective data from Lucas
et al. (2010) showed that patients with bronchiectasis require twice as much replace-
ment doses to achieve the same IgG level compared with those free of bronchiectasis.
Prospective trials, however, addressing this question, have never been performed.
For further details of replacement therapy, I would like to refer to existing

national and international guidelines for the use of immunoglobulins in antibody-
deficient patients.
2 From Immune Substitution to Immuno-modulation 17
2.3 Immunomodulation in Autoimmune Diseases
The second milestone in the history of the clinical use of immunoglobulins was
the publication by Imbach et al. (1981). The authors described seven children with
chronic and six with acute ITP in whom the platelet count was markedly increased
through high-dose administration of immunoglobulins. The course was variable,
with some patients needing IVIG infusions on a regular basis. Several publica-
tions by other authors confirmed these initial observations in controlled clinical
trials.
As Imbach’s paper had shown that certain immunopathological reactions may
be modified by IVIG, it encouraged many authors worldwide to exploit the poten-
tial of IVIG treatment in other diseases too. The number of diseases where this
mode of treatment was attempted may now exceed 100. Not all attempts were suc-
cessful, and only a few turned out to be “indications” on the basis of randomized
controlled trials (RCT). However, in some very rare diseases, the call for RCT may
be inadequate because the number of patients is simply too small. Keeping this in
mind even case reports may be meaningful. Many results and “indications” are
summarized in a small booklet (Wahn and Orange 2013) and in a recent review
(Perez et al. 2017).
Imbach et al. (1981) used a 7S preparation of IV gamma globulin (Sandoglobulin),
which had an intact Fc fragment. Later publications showed the poorer effect of
F(ab′)2 fragments (=5S IgG) (Burdach et al. 1986). In contrast, a good clinical
effect could be achieved with purified Fc fragments devoid of F(ab′)2 (Debré et al.
1993). A substantial part of the biological effect of IgG is thus dependent on the
presence of the Fc portion suggesting that the interaction of IVIG with the various
types of Fc receptors is crucial for its efficacy (Fig. 2.1).
FcR Blockade by IgG
Plt
Plt Binding to
IgG Fc receptor
Binding to blocked
Fc receptor by IgG
possible
Mϕ
Fig. 2.1 In ITP, induced by known or unknown triggers autoantibodies bind to platelets which are
taken up by cells of the reticuloendothelial system via Fc receptors expressed at the surface. If such
receptors are blocked my therapeutic IgG, antibody-coated platelets remain in the circulation
18 V. Wahn
Beyond these interactions many other mechanisms may be effective either alone
or in concert which have been discussed in detail (Imbach et al. 2010; Matucci et al.
2015).
2.4 Immunomodulation in Alloimmune Diseases
After having studied Imbach’s work, my colleagues and I were inspired to study the
potential role of IVIG in another pediatric disease affecting newborn babies, rhesus
hemolytic disease. The pathomechanism of disease can be illustrated as follows
(Fig. 2.2):
If so we hypothesized that IVIG could decrease the degree of hemolysis and thus
reduce the number of exchange transfusions required, the first three cases (Rübo
and Wahn 1990) suggested that, in fact, IVIG modified the course of bilirubin
incline and allowed us to avoid exchange transfusions. This observation motivated
us to study the effects of IVIG in a larger group of babies in a randomized controlled
trial (Rübo and Wahn 1991; Rübo et al. 1992). In this setting, we were able to show
that the number of exchange transfusions could be significantly reduced if IVIG was
given early enough in babies at risk for bilirubin encephalopathy.
As IVIG does not reduce the number of antibody-coated red blood cells but only
slows down their uptake by the RES, we followed all children for the development
of late anemia. In fact, a few IVIG-treated babies required blood transfusions for
late anemia. Because, however, the risk of a blood transfusion is by far lower than
the risk of an exchange transfusion, we considered this risk acceptable.
Our observation has been confirmed in later publications reporting results in babies
with rhesus and ABO hemolytic disease. In 2004, the American Academy of Pediatrics
recommended prophylactic IgG as an alternative for blood exchange transfusions.
Rhesus Hemolytic Disease - Pathogenesis
Rh
Fig. 2.2 During
pregnancy, fetal rhesus D+
red blood cells stimulate
Pre-and postnatal
the generation of maternal hemolysis
anti-Rh D alloantibodies. Rh
These can cross the

placenta and may destroy
Rh
Rh D+ red blood cells in
the baby by Fc receptor
mediated uptake by cells of
the reticuloendothelial
system. This is the major
mechanism as anti-D
antibodies do not bind
complement
2.5 Immunomodulation in Inflammatory Diseases
While the two previous examples are associated with disease-causing specific anti-
bodies, the experiences in Kawasaki disease (KD; for review see Agarwal and
Agrawal 2017) expanded the spectrum of IVIG efficacy to a pediatric vasculitis
with unknown etiopathogenesis but with massive proinflammatory cytokinemia.
The disease may be based on a complex interplay of genetic factors, infections, and
immunity. KD mainly affects infants and toddlers. The major problem is the devel-
opment of coronary artery aneurysms which are responsible for fatal courses.
Initially, the disease was treated with aspirin at high doses. However, despite such
treatment, aneurysms still occurred, and new modes of treatment had to be
developed.
Keeping this background in mind, the paper by Newburger et al. (1986) for the
first time showed that IVIG had an anti-inflammatory potential. In randomized
trial, aspirin alone was compared to the combination of aspirin + IVIG at a dose
of 4 × 400 mg/kg bw given on four consecutive days. Combination therapy
reduced the number of coronary artery aneurysms significantly after 2 (from 23 to
8%) and after 7 weeks (from 18 to 4%). In a subsequent trial (Newburger et al.
1991), the administration of 2 g/kg bw given on 1 day compared to 4 × 400 mg/kg
bw on 4 days further reduced the risk for aneurysms by another approximately
50%. Since then 2 g/kg bw is an effective treatment for most of the kids. If symp-
toms persist based on data from appropriate trials, German guidelines recommend
the addition of oral steroids. For resistant cases German Guidelines recommend
infliximab.
The mode of action of IVIG is not quite clear. Maybe that like in ITP several
mechanism may act in concert. The following figure illustrates only one of the
mechanisms described in the literature (Fig. 2.3).
Possible Mechanism:
Neutralization of Superantigens
T cell
α β Staphylococcal
enterotoxin B
A peptide V J J D V
activates
Pep Staphylococcal
a few
enterotoxin B
T cell only MHC
A
superantigen
APC activates a
Staphylococcal
whole
enterotoxin B
Vβ family IVIG
Fig. 2.3 In specific T-cell responses the antigen-presenting cell presents peptides which are rec-
ognized by a single clone of antigen-specific T cells using α- and β-chain of the T-cell receptor. In
KD the so-called superantigens expressed by certain bacteria may activate a whole Vß family of T
cells leading to a massive cytokine response. IVIG may neutralize these superantigens and, thus,
reduce the inflammatory response
20 V. Wahn
Whatever the mechanism of disease and the mode of action of IVIG may be,
the fact remains that in addition to some autoimmune and alloimmune disorders
also some disorders characterized by massive inflammation can be influenced
by IVIG.
2.6 Expansion to New Indications
Inspired by the fascinating chance to modify immunopathology by IgG as a kind of

treatment associated with a relatively low-risk clinicians worldwide looked for pos-
sible further indications. Figure 2.4 tries to illustrate that.
Not all attempts were successful. However, some clear indications have emerged
making IgG treatment indispensable in clinical practice.
Immunomodulation with IgG:

From ITP to other Applications
Neurology
Hematology Rheumatology
ITP
Dermatology Organ Tx
Gynecology
Fig. 2.4 The efficacy of IgG treatment as a mode to modify abnormal immune responses was first
demonstrated in children with ITP. Since the several other potential applications have emerged
(summarized in Wahn and Orange (2013) and Perez et al. (2017))
Disclosures In the last years, the author has received honoraria for scientific lectures from
Octapharma, CSL Behring, Biotest, PPTA and the FIND-ID network. He was also paid for his
work in an advisory board (Pharming) and data safety monitoring board (Octapharma, Pfizer).
References
Agarwal S, Agrawal DK. Kawasaki disease: etiopathogenesis and novel treatment strategies. Exp
Rev Clin Immunol. 2017;13(3):247–58.
Bonagura VR, Marchlewski R, Cox A, Rosenthal DW. Biologic IgG level in primary immunodefi-
ciency disease: the IgG level that protects against recurrent infection. J Allergy Clin Immunol.
2008;122(1):210–2.
Bruton OC. Agammaglobulinemia. Pediatrics. 1952;9(6):722–8.
Burdach SE, Evers KG, Geursen RG. Treatment of acute idiopathic thrombocytopenic purpura of
childhood with intravenous immunoglobulin G: comparative efficacy of 7S and 5S prepara-
tions. J Pediatr. 1986;109(5):770–5.
Debré M, Bonnet MC, Fridman WH, Carosella E, Philippe N, Reinert P, Vilmer E, Kaplan C,
Teillaud JL, Griscelli C. Infusion of Fc gamma fragments for treatment of children with acute
immune thrombocytopenic purpura. Lancet. 1993;342(8877):945–9.
Imbach P, Barandun S, d’Apuzzo V, Baumgartner C, Hirt A, Morell A, Rossi E, Schöni M, Vest M,
Wagner HP. High-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura
in childhood. Lancet. 1981;1(8232):1228–31.
immunomodulations in autoimmune disorders. Vox Sang. 2010;98(3 Pt 2):385–94.
Janssen WJ, Mohamed Hoesein F, Van de Ven AA, Maarschalk J, van Royen F, de Jong PA, Sanders
EA, van Montfrans JM, Ellerbroek PM. IgG trough levels and progression of pulmonary dis-
ease in pediatric and adult CVID patients. J Allergy Clin Immunol. 2017;140(1):304–306.e4.
Lucas M, Lee M, Lortan J, Lopez-Granados E, Misbah S, Chapel H. Infection outcomes in patients
with common variable immunodeficiency disorders: relationship to immunoglobulin therapy
over 22 years. J Allergy Clin Immunol. 2010;125(6):1354–60.
Matucci A, Maggi E, Vultaggio A. Mechanisms of action of Ig preparations: immunomodulatory
and anti-inflammatory effects. Front Immunol. 2015;5:690.
Newburger JW, Takahashi M, Burns JC, Beiser AS, Chung KJ, Duffy CE, Glode MP, Mason WH,
Reddy V, Sanders SP, et al. The treatment of Kawasaki syndrome with intravenous gamma
globulin. N Engl J Med. 1986;315(6):341–7.
Newburger JW, Takahashi M, Beiser AS, Burns JC, Bastian J, Chung KJ, Colan SD, Duffy
CE, Fulton DR, Glode MP, et al. A single intravenous infusion of gamma globulin as com-
pared with four infusions in the treatment of acute Kawasaki syndrome. N Engl J Med.
1991;324(23):1633–9.
Orange JS, Grossman WJ, Navickis RJ, Wilkes MM. Impact of trough IgG on pneumonia inci-
dence in primary immunodeficiency: a meta-analysis of clinical studies. Clin Immunol.
2010;137(1):21–30.
Perez EE, Orange JS, Bonilla F, Chinen J, Chinn IK, Dorsey M, El-Gamal Y, Harville TO, Hossny
E, Mazer B, Nelson R, Secord E, Jordan SC, Stiehm ER, Vo AA, Ballow M. Update on the
use of immunoglobulin in human disease: a review of evidence. J Allergy Clin Immunol.
2017;139(3S):S1–S46.
22 V. Wahn
Rübo J, Wahn V. A trial with high-dose gamma globulin therapy in 3 children with hyperbilirubi-
nemia in rhesus incompatibility. Monatsschr Kinderheilkd. 1990;138(4):216–20.
Rübo J, Wahn V. High-dose intravenous gammaglobulin in rhesus-haemolytic disease. Lancet.
1991;337(8746):914.
Rübo J, Albrecht K, Lasch P, Laufkötter E, Leititis J, Marsan D, Niemeyer B, Roesler J, Roll C,
Roth B, et al. High-dose intravenous immune globulin therapy for hyperbilirubinemia caused
by Rh hemolytic disease. J Pediatr. 1992;121(1):93–7.
Wahn V, Orange JS. Clinical use of immunoglobulins UNI-MED science. 2nd ed. Bremen: UNI-
MED Verlag AG; 2013.
Manual of Primary and Secondary
Immunodeficiencies 3
Paul Imbach and Volker Wahn
3.1 Introduction
This chapter focuses on practical issues of management. A short summary presents

the characteristics of the distinct disorders (for details see reviews).
Replacement with human polyclonal immunoglobulin concentrate is one of the
main modes of treatment for the majority of patients with primary immunodeficien-
cies (PID). PID may present with life-threatening or severe infections, autoimmune
diseases, lymphoproliferation, and malignancies. Many deficiencies are associated
with impaired antibody production in addition to dysfunction of other components
of the immune system.
To date, over 300 monogenetic PID have been described in the literature. Here,
only a few classical examples are characterized.
3.2 X-Linked (Bruton’s) Agammaglobulinemia
3.2.1 Definition and Prevalence
X-linked agammaglobulinemia is an autosomal recessive disorder characterized by

severe reduction of plasma immunoglobulins and absence of B cells. The preva-
lence is estimated at 1:100,000.
P. Imbach
University of Basel, CH 4031, Basel, Switzerland
V. Wahn (*)

24 P. Imbach and V. Wahn
3.2.2 Pathophysiology
The disease is caused by various mutations of BTK (Bruton’s tyrosine kinase)

which results in the absence of mature B lymphocytes, profound reduction of all
classes of Immunoglobulins, and, lastly, the absence of antibody production.
Precursor B cells (PreB) remain present in the bone marrow.
3.2.3 Clinical Manifestations
Recurrent infections: pneumonia, sinusitis, otitis, later on bronchiectases and gastro-

intestinal disorders, arthritis, viral meningoencephalitis, and autoimmune diseases.
After live vaccination with oral polio vaccine: vaccine-derived poliomyelitis.
Lymphatic tissue (tonsils, lymph nodes) small or absent.
3.2.4 Diagnostics
IgG, IgA, IgM, and IgE absent or very low. Absence of specific antibodies (tetanus,
pneumococci) despite regular vaccinations. Absence of CD19/CD20 positive B
cells. Typical mutations in the BTK gene.
3.2.5 Differential Diagnosis
Severe combined immunodeficiency (SCID, see 3.3), transient hypogammaglobu-

linemia of infancy.
Hypogammaglobulinemia due to loss of IgG (enteric, renal).
3.2.6 Therapy
IV- or SC-IgG substitution to obtain a plasma level >8 g/l IgG, usually achieved by
doses of 0.3–0.6 g IgG/kg body weight every 3–4 weeks. Patients with bronchiecta-
sis may require higher doses.
Aim of treatment: prevention of severe infections, survival, and improved quality
of life.
3.2.7 Prognosis
With IgG substitution from early life onwards: normal to slightly reduced life expectancy
Without substitution: fatal infections in childhood
3 Manual of Primary and Secondary Immunodeficiencies 25
3.3 Severe Combined Immunodeficiency (SCID)
3.3.1 Definition and Prevalence
Combined cellular and humoral immunodeficiency with absent T cells and absent
or dysfunctional B cells. Prevalence: 1:50,000 (USA). Without treatment, usually
fatal within the first year of life.
3.3.2 Pathophysiology
3.3.2.1 Different Forms
T-B + Variants
Several mutations in genes for cytokine receptors, subunits of the T-cell receptor
complex, other membrane proteins, signal transduction molecules and coronin 1A
(necessary for thymic release of mature T cells).
T-B-Variants
Several mutations in genes responsible for stem cell maturation, B-/T-cell DNA
recombination, and purine metabolism (ADA)
–– Severe recurrent and opportunistic infections within the first months of life.
–– Chronic graft-versus-host disease caused by maternal T cells, severe CMV, bac-
terial and fungal infections, Pneumocystis jiroveci pneumonia, chronic diarrhea
and/or failure to thrive, and complicated infections by live vaccines (rotavirus,
BCG)
3.3.4 Diagnostics
–– Reduced lymphatic tissue, i.e., lateral thorax X-ray with small or absent thymus
in some genetic variants
–– FACS: Absence of T cells, some variants also lack B cells. Atypical findings with
maternal engraftment, Omenn phenotype, or leaky SCID variants
–– Serum IgG, IgM, IgA all markedly reduced
–– Impaired T-cell proliferation with mitogens and antigens, absent antibody pro-
duction after vaccinations
3.3.5 Treatment
Sterile environment, avoidance of live vaccines, avoidance of non-irradiated blood

transfusions
–– Antibacterial, antifungal, and antiviral treatment of infections

–– IgG substitution as under A as long as B cells are not reconstituted by stem cell
transplantation
–– Parenteral nutrition if required
–– Stem cell transplantation from matched family or unrelated donor. Haploidentical
Tx less effective
–– Somatic gene therapy only approved for ADA deficiency in cases where a
matched family donor is not found
3.4 Hyper-IgM Syndromes
3.4.1 Pathogenesis
–– Antibody class-switch defects: (a) X-chromosomal, recessive type with gene

mutation of CD40 ligand (CD40L) and (b) autosomal recessive types (CD40
deficiency, uracil N-glycosylase deficiency (UNG), activation-induced cytidine
deaminase (AID) deficiency, and several others)
–– Presence of B cells, but low serum levels of IgG and IgA and normal or high
levels of IgM
3.4.2 Clinical Presentation
CD40 and CD40L deficiencies are regarded as combined B-/T-cell deficiencies,

while UNG and AID are predominantly B-cell deficiencies.
–– Bacterial respiratory infections

–– Pneumocystis jiroveci infection (CD40 and CD40L), especially in the first year of life
–– Diarrhea, infections with cryptosporidium
–– Lymphadenopathy, neutropenia, thrombocytopenia, anemia
3.4.3 Treatment
–– Treatment of existing infections

–– IgG substitution as under Sect. 3.2.6
–– Stem cell transplantation in selected patients with CD40 or CD40L deficiencies
3.5 Common Variable Immunodeficiency (CVID)
3.5.1 Incidence and Diagnosis
Common variable immunodeficiency (CVID) is the most frequent symptomatic

PID. 10–20% are monogenetic disorders, some with autosomal dominant and some
with autosomal recessive inheritance. Estimated prevalence 1:10,000–1:50,000.
(a) Reduction of IgG and IgA

(b) IgM may be normal
(c) Impaired antibody response to vaccines
(d) Other causes excluded
The diagnosis can be made in the absence of recurrent infections if (a)–(d) are
met (according to International Consensus 2016).
–– Infections similar to agammaglobulinemia (A.1), development of bronchiectasis,

and chronic lung disease
–– Noninfectious manifestations: diarrhea, malabsorption, high rate of autoimmune
disorders such as rheumatoid arthritis, autoimmune hemolytic anemia, thrombo-
cytopenia, neutropenia granulomatous and lymphoproliferative complications,
malignancies
3.5.3 Treatment
–– IgG substitution as in Sect. 3.2.6
Treatment of infections
Immunosuppression for autoimmune and granulomatous complications
Stem cell transplantation is not a standard treatment
3.6 Selective Antibody Deficiency
3.6.1 Definition and Diagnosis
–– Immunoglobulins IgG, IgA and IgM normal, B cells normal

–– Impaired vaccine antibody production to pneumococcal polysaccharide vaccine
(Pneumovax®)
–– Recurrent infections with encapsulated bacteria
3.6.2 Treatment
–– Antibiotic prophylaxis and/or treatment

–– IgG substitution as Sect. 3.2.6 in severe cases
3.7 Transient Hypogammaglobulinemia of Infancy
3.7.1 Definition and Diagnosis
–– Delay of B-cell maturation

–– Affects mainly infants and toddlers <6 years of age
–– Usually no abnormal susceptibility to infections
–– Hypogammaglobulinemia with normal antibody responses
3.7.2 Management
–– Usually no IgG substitution required
3.8 Isolated IgG 1–3 Subclass Deficiency
–– Some patients with recurrent infections

–– Minority with deficient antibody response to polysaccharide vaccines, especially
in the absence of IgG2
3.8.1 Management
–– Antibiotic treatment of infections

–– IgG-substitution when polysaccharide responses are absent
3.9 elective IgA Deficiency with or without IgG2

S
Deficiency
–– Selective IgA deficiency usually asymptomatic, slightly increased risk for celiac
disease, rheumatic diseases and allergies
–– Increased susceptibility to infections if associated with IgG2 deficiency
–– In rare cases, risk of anaphylactic reaction to IgG substitution or blood transfu-
sions due to the presence of autoantibodies to IgA
–– If IgG substitution is indicated: use of IgG-preparation with low IgA content
3.10 Wiskott-Aldrich Syndrome (WAS)
3.10.1 Pathogenesis/Etiology
–– X-chromosomal inheritance
–– Combined immunodeficiency with high levels of serum IgE and IgA and low
serum IgM, impaired polysaccharide responses
–– Defective protein (WASP) leads to impaired signal transduction and actin
poly-merization following lymphocyte activation
–– Thrombocytopenic bleeding, eczema, recurrent infections, and increased risk for

malignancies
3.10.3 Treatment
–– Administration of IVIG, antibiotic, and antiviral treatment.

–– Allogenic stem cell transplantation may be curative.
–– Somatic gene therapy is still experimental.
3.11 Ataxia Telangiectasia
3.11.1 Pathophysiology/Etiology
–– Autosomal recessive disorder with ATM-gene mutations. ATM is involved in

repair of DNA strand breaks and control of cell cycle. Radiosensitive disorder
with chromosomal instability.
–– Some patients present with combined ID, variable expression of hypogamma-
globulinemia, low IgG subclasses, low IgA and IgE, low antibody responses.
–– Some patients have a hyper-IgM phenotype. Alpha-1 fetoprotein usually
elevated.
3.11.2 Clinical Manifestation
–– Susceptibility to all kinds of infections. Development of telangiectases and cer-

ebellar ataxia in the first year of life, later on high risk for lymphatic and other
malignancies
3.11.3 Treatment
–– Treatment of infections
–– IgG substitution may be useful if severe infections occur and specific antibody
responses are markedly impaired
–– Prognosis is still poor
3.12 Secondary Immunodeficiency
3.12.1 C
hronic Lymphocytic Leukemia (CLL), Multiple Myeloma (MM)
and Treatment of Related Secondary
Immunodeficiency in Patients with Hematologic or
with Solid Tumor Malignancy with or without
Transplantation
General Aspects
–– Due to novel treatment regimens with immunosuppressive, anticancer drugs,

anti-inflammatory drugs, and monoclonal antibodies, which have significantly
prolonged malignancy-related survival and also increased the risk of morbidity
and mortality due to severe infection and the underlying diseases, the indication
of IVIG has to be reconsidered.
–– The cumulative treatments lead to immune defects such as severe neutropenia,
mucosal lesions, T-cell dysregulations, natural killer cell dysfunction, cytokine
alterations, phagocytic dysfunctions, complement activation, apoptotic changes,
and many other immune response alterations. In addition, hypogammaglobu-
linemia due to defective B-cell production of polyvalent antibodies may be
present.
–– The combined treatment regimen with monoclonal antibodies such as alemtu-
zumab, anti-CD20, chimeric antigen receptor (CAR) T cells, Bcl-2 antagonist,
tyrosine kinase inhibitor ibrutinib, Syk inhibitor fostamatinib, and others increase
the risk of reactivation of herpes virus, hepatitis B infections, the risk of oppor-
tunistic infections, such as Pneumocystis jiroveci, listeria, mycobacteria, and
candida, and the risk of encapsulated bacteria (Staphylococcus pneumoniae,
Staphylococcus aureus, Haemophilus influenzae) infection/pneumonia and
bronchiectasia.
–– Since the majority of patients with these diagnoses have secondary immunodefi-
ciency, analyses are recommended prior to, during, and after specific treatment,
namely, history of infections and vaccinations, clinical examinations, differential
blood analyses, FACS including CD4 and CD8 T cells and B cells, and quantita-
tive analysis of serum IgG with subclasses, IgM, IgA, and IgE, antigen-specific
antibody production (e.g., to polysaccharide antigens (T-cell-independent
response) as by the 23-valent pneumococcal vaccines), and antibody responses
to tetanus toxoid or influenza virus.
Supportive Management
–– Vaccinations (all live vaccinations are contraindicated!) before specific treatment

(if possible) and after completion of treatment, when indicated by the results of
analyses (see above).
–– IVIG replacement as prophylaxis and treatment in selected patients with defec-
tive immunity and those with recurrent infection refractory to or insufficiently
responding to antibiotics/antiviral/antifungal treatment.
–– IVIG may also influence the defense against the underlying disease.
–– Dosage of IVIG: As in primary immune deficiencies with the aim to reach a
serum IgG level of 6–8 g/l, as long as necessary/as recovery of the immunologi-
cal functions.
3.12.2 Specific Aspects
3.12.2.1 CLL and MM

• In the past, patients with CLL, hypogammaglobulinemia, and recurrent infec-
tions have been significantly protected from infections by administration of IVIG
in several controlled studies, it rarely is used, today with the conventional
treatment.
• For new combined treatment see “Gemeral aspects” above.
3.12.2.2 Hematopoietic Stem Cell Transplantation HSCT

• Currently, IVIG prevention treatment of graft-versus-host disease (GVHD) and
infection during the peritransplantation time is no longer recommended in
patients with HLA-identical HSCT except in patients with severe secondary
immunodeficiency/complications (see above).
• Patients with HSCT due to severe combined immunodeficiency and other pri-
mary or secondary immunodeficiencies with a- or hypogammaglobulinemia are
candidates for IgG preventive treatment. Additionally, infants should be substi-
tuted by IVIG before and after HSCT as long as reconstitution of the immune
functions is established. Thus, individual IgG administration is indicated in these
groups of patients.
3.12.2.3 Solid Organ Transplantation

• IgG treatment is beneficial for sensitized patients exposed to ABO blood group
antigens, including from platelet transfusions, during the wait time for organ
transplantation, i.e. kidney transplantation, and for patients after non-identical
HLA antigen exposure due to transplantation.
• High dose IVIG administration with or without monoclonal B-cell depletion (i.e.
rituximab) and/or plasma exchange reduces the level of anti-HLA antibodies and
improves the rate of transplantation.
• IVIG and monoclonal B-cell depletion (i.e. rituximab) can be useful for desensi-
tization, especially before, during and after allogenic heart and/or lung transplan-
tation due to donor-specific HLA antibodies. Donor-specific HLA antibodies are
an important risk factor of bronchiolitis obliterans.
• In patients with antibody-related rejection of a transplantation, the combination
of IVIG, monoclonal B-cell depletion (i.e., rituximab), and eventually plasma
exchange is the recommended approach.
• Patients during and after non-HLA-identical or haploid HSCT may benefit from
IgG preventive treatment for a limited time, i.e., until reconstitution of the
immune functions.
3.12.3 HIV Infection in Children
• IgG substitution has shown positive effects for HIV infected children with CD4
T cells >200/μl in controlled clinical studies. However, these studies have been
performed before highly active antiviral treatment (HAART) became available.
Today, children with HIV infection treated with HAART show reconstitution of
T cells and normalization of antibody responses. Thus, IgG administration in
HIV infected children is an approved indication which is no longer practiced.
3.12.4 Preterm Infants
• IgG substitution in premature babies has been studied in many trials using a
prophylactic or therapeutic design. Based on the latest large trials, it became
clear that babies do not benefit from IgG treatment.
3.12.5 Geriatrics: Immunosenescence
• Elderly people may have deficient adaptive and innate immune responses.
• Evaluation of the immune state and function is indicated, if recurrent, severe, or
difficult to treat infections are present, i.e., by anomalies of FACS, IgG, and sub-
classes, antibody responses to vaccines.
• IgG substitution may be considered in elderly patients with immune deficiencies.
Dosage as in Sect. 3.11.3.
• Adverse effects to IgG treatment see Chap. 10 are higher in patients over 60 years
of age.
• Serious adverse effects in older patients include higher risk of acute, transient
renal failure and venous thrombosis depending on comorbidities.
• Recommendation of administration of IVIG in elderly patients: sufficient hydra-
tion prior to IVIG, slow infusion rate, IVIG preparation with low concentration
of sucrose, and monitoring renal function.
• In the future: subcutaneous Ig SCIG administration may have less adverse
effects.
3.13 Other Combined Immunodeficiency
• Among the over 300 monogenetic disorders leading to immunodeficiency, there

are many with impaired antibody responses where affected patients benefit from
IgG replacement. Several of these combined ID, however, can be corrected by
stem cell transplantation. If thereafter the B-cell compartment is fully reconsti-
tuted, IgG administration may become dispensable.
Manual of Intravenous
and Subcutaneous IgG Indications 4
in Autoimmune Diseases
Paul Imbach
4.1 Introduction
This manual is a brief summary of extensive reviews of the literature extracted from
PubMed and a recent review by Perez et al. (2017), which mainly categorizes the
indications according to FDA/EMA approvals and evidence-based findings.
The manual is written by a physician involved in the clinical care of patients
considering the different individual manifestations and the complex characteristics
of the autoimmune and inflammatory diseases. It focuses on the clinical options of
recommended therapeutic, immunomodulatory effects of administration of IVIG
and categorizes them in respect to other treatment approaches.
4.2 General Characteristics of Autoimmune Disorders
Autoimmune disorders are characterized by:
• Clinical manifestations and follow-ups of inflammatory and autoimmune dis-

eases are heterogenous and variable.
• The disorders are often associated with pathogenic antigen(s), antibodies, or
immune complexes.
• The immune system is imbalanced, and the innate and/or adaptive immune
responses differ from patient to patient.
• A minority of common autoimmune diseases, where controlled (randomized
and/or blinded) clinical studies could be performed leading to evidence-based
recommendation (category A, see below).
P. Imbach
University of Basel, CH 4031, Basel, Switzerland

36 P. Imbach
• The majority of autoimmune disorders which are rare and controlled clinical
studies often are not feasible; therefore, rare inflammatory or autoimmune dis-
eases are off-label indications of IVIG (category B, see below).
• Continuous discussion and assessment of each indication of administration of
intravenous or subcutaneous immunoglobulin (IVIG or SCIG) concentrate are
necessary.
4.3 eneral Characteristics of IVIG

G
as an Immunomodulatory Biological Agent
In contrast to substitutive administration of IVIG for primary and secondary immu-

nodeficiencies, immunomodulatory administration of IVIG is characterized:
• By high dosage of IVIG usually 2 g/kg within 1–5 days per course (named HD
IVIG in the following texts); for other dosage recommendations and for the new
possibility of SCIG administration, see text of the respective disorder.
• By adverse effects of HD IVIG which are mild or moderate at a rate of 5%;
however, some severe side effects are known.
• By the high demand of IVIG, and this limits the potential indications because of
the shortages, of the costs, etc.
• By the immunomodulatory mechanisms of action of IVIG, which involves the
whole innate and adaptive immune response in a synergistic and complex way
(Imbach et al. 2010) and, therefore, remains unclear and often unexplained.
• By the health authorities, and the insurance providers who may disagree on cov-
ering the cost of this biological agent for certain indications.
4.4 linical Categorization of IVIG Indications

C
of Autoimmune and Inflammatory Disorders
In the above respect, the following categorization of the various IVIG indications
will be used:
• According to evidences, guidelines, and literature reviews/updates

• According to the need of individual indications due to efficacies; due to the fail-
ure of other treatments, which may induce secondary immunodeficiency or other
unacceptable adverse effects or which may be contraindicated; due to comorbidi-
ties, or as an adjuvant to first-line treatment, etc.: For details: See Table 4.1
• Always keeping in mind that indications may change by development of new
biological drugs (e.g., monoclonal antibodies) or by new recognition of the com-
plex pathophysiology of the diverse diseases
The following categories are indicated prior to each below-mentioned indication

(Suggestion to the reader; Copy table 4.1 for your convenience):
4 Manual of Intravenous and Subcutaneous IgG Indications in Autoimmune Diseases 37
Table 4.1 Categories for the below mentioned indications

Aa Evidence due to controlled (randomized and/or blinded) trials and approved by
authorities
Ab Guidelines/recommendations by experts/committees and reviews/updates
(e.g. Cochrane, others)
Ac Association with primary/secondary immune deficiencies
Ba Efficacies in individual patients or cohorts with very rare diseases with any
possibilities of reliable clinical studies
Bb Efficient alternative
– to other treatments,
– to few or no treatment alternatives,
– to contraindications of drugs with adverse effects,
– with drug sparing effects (e.g. corticosteroids, other immunosuppressives)
Bc Adjuvant treatment in combination with other drugs (e.g. corticosteroids, other
immunosuppressive drugs, monoclonal antibodies)
Bd Failure of conventional/first line treatment (e.g. corticosteroid, other
immunosuppressants, antiepileptics)
Be High fatality of a disease
Bf Relevance of improvement of quality of life
C Historical studies with some specific, efficient aspects, but insufficient evidence
D Documentation of any efficacies
4.5 Hematology
4.5.1 A, a* Immune Thrombocytopenia (ITP) (*See Table 4.1)
• IVIG prevents or fastly controls patients with bleedings.

• This disorder was the “door opener” for the immunomodulatory effects of IVIG
(see Chap. 1 and Part III).
4.5.2 A, a* Alloimmune Thrombocytopenia (*See Table 4.1)
1. A, a *Fetomaternal alloimmunization (*See Table 4.1)

• Rare disease, high risk of intraventricular, fatal hemorrhages.
• Maternal alloantibodies against platelet antigen HPA-1a (responsible for 80%
of fetomaternal alloimmunization).
• Recurrence rate during subsequent pregnancy is 79%.
• Diagnosis: paternal genotype of HPA-1a involved in the preceding fetomater-
nal alloimmunization; when paternal heterogeneity is present, maternal
incompatibility must be analyzed by cell-free fetal DNA.
• Treatment: no longer invasive fetal analyses (fetal blood sample) or treatment
(intrauterine fetal platelet tr\ansfusions); complication rate is 11%.
• 2 × 1 g IVIG/week to the mother, repetitions weekly, starting at the 20th week
of gestation.
38 P. Imbach
• Prognosis: 3% intracranial hemorrhage (retrospective result of n = 839, and

4% mortality of n = 821).
(Winkelhorst et al. 2017; Rayment et al. 2005, 2011)
2. A, a* Postnatal alloimmune thrombocytopenia (*See Table 4.1)

• IVIG administrations in neonates until platelet counts remain <30 × 109/l.
3. A, a* Rhesus hemolytic anemia (*See Table 4.1)
• Fetal Rhesus D+ red blood cells stimulate maternal anti-Rh D alloantibodies,
which cross the placenta and destroy Rh D+ blood cells in the baby.
• IVIG modifies the course of bilirubin incline and significantly reduces the
number of exchange transfusions and decreases the risk of bilirubin
encephalopathy.
(Rübo et al. 1992, see also Chap. 3 in this book.)
4. B, a, c* Posttransfusion purpura (*See Table 4.1)

• Thrombocytopenia occurs 7–10 days after transfusion of platelet-containing
blood products with positive platelet antigen 1 or anti-HLA class 1 to a patient
with negative antigen 1 or anti-HLA class 1 antigen, which may cause life-
threatening bleeding.
• Treatment: HD IVIG, individually or together with IV corticosteroids, plas-
mapheresis, and/or rituximab.
(Mueller-Eckhardt and Kiefel 1988)
4.5.3 B
, a* Pure Red Cell Anemia Associated with Chronic
Parvovirus B19 Infection (*See Table 4.1)
• Rare, severe anemia (<7 g/l hemoglobin).

• Occurs often, but not always in immunocompromised patients (HIV infec-
tion, posttransplantation, primary immunodeficiency, hematologic
malignancy).
• Treatment: HD IVIG, corticosteroid, cyclosporine A, rituximab (in patients with
associated disease).
• Prognosis: response rate to first course of IVIG is 93%; relapse rate is 33.9%
within 4.3 months.
(Crabol et al. 2013)

4.5.4 B
, a, e* Thrombotic Thrombocytopenic Purpura (TTP)
(*See Table 4.1)
• Rare microangiopathic coagulopathy, observed in patients with mutations of

ADAMTS13 or with para-/postinfectious and immune dysregulated conditions.
• Prognosis may be fatal.
• Treatment: daily plasma exchange, HD IVIG, corticosteroids, rituximab, and
immunosuppressive drugs.
(Moore et al. 2007)
4.5.5 B, a, c* Autoimmune Neutropenia (*See Table 4.1)
• Severe form: ANC <0.5 × 109/l.

• Pathogenesis: autoantibodies against neutrophils, spontaneous recovery
possible.
• First-line treatment: G-CSF, GM-CSF.
• In patients with infection or immunodeficiency including patients after stem cell
transplantation, HD IVIG as adjuvant treatment is effective.
(Marriotti et al. 2014) See comment in PubMed Commons below (Marriotti et al.
2014).
4.5.6 B
, a, d* Autoimmune Hemolytic Anemia (AIHA) and Evans
Syndrome (affecting 2–3 Hematopoietic Lineages)
(*See Table 4.1)
• AIHA is a rare disease (1–3:100,000 people per year).

• Autoantibodies against red blood cells.
• Classified as warm (wAIHA), cold (CAD), or mixed/paroxysmal form depend-
ing on the thermal range of autoantibodies. It may be as secondary AIHA associ-
ated with other concomitant disorders such as infection, immunodeficiencies,
lympho- or myeloproliferative disorder or cancer, and drugs.
• Evans syndrome (±35%) is an autoimmune destructive disorder of 2–3 hemato-
logic lines (mostly erythrocytes and thrombocytes).
• Diagnosis is based on hemolysis, detectable by the direct antiglobulin test (DAT):
–– Warm AIHA (wAIHA) by anti-IgG antisera (Cd3) (rate 65–70%/).
–– Cold AIHA (CAD) by IgM autoantibodies/Cd3 agglutinating at 37 °C (rate
20–25%).
40 P. Imbach
–– The third form binds IgG autoantibodies to erythrocytes in the cold, but not at
37 °C, named paroxysmal cold hemolysis due to Donath-Landsteiner anti-
bodies (rate 1–3%).
• First-line treatments are corticosteroids, rituximab or other immunosuppressive
drugs, splenectomy (in adults with severe wAIHA only), or plasma exchange:
–– CAD: avoidance of cold; if symptomatic (hemolysis), rituximab as mono-
therapy or in combination with fludarabine
–– Evans syndrome: IVIG, corticosteroids; when refractory, rituximab
• HD IVIG is frequently administered in children with wAIHA due to less adverse
effects and benefits with or without immunosuppressants.
• Prognosis: 40–64% complete recovery, 30–40% chronic/relapsing forms, and
4% mortality in case series.
(Liebman and Weitz 2017)
4.5.7 B, a, c* Acquired Hemophilia (*See Table 4.1)
• Rare autoimmune disorder with severe, life-threatening bleeding.

• Inhibitory autoantibodies against coagulation factor, mainly directed against
FVIII.
• Occurs in postpartum patients, with connective tissue disease, with p araneoplastic
syndrome, and drug and herbal related.
• Spontaneous recovery within 14 months is possible.
• Treatment:
–– Emergency: FVIII replacement or recombinant FVII administration; new:
emicizumab activating FIX and FX with longer half-life (administration weekly)
–– Long-term treatment: immunosuppressive drugs; in refractory patient, HD
IVIG or a combination of immunosuppressants and HD IVIG
(Mo and Bao 2017)
4.5.8 B
, a, c* Acquired Autoimmune Coagulation Factor
Inhibitors and Acquired von Willebrand Syndrome
(*See Table 4.1)
• Common hereditary form and rare acquired form, mainly associated with lym-
pho- or myeloproliferative disorders with prolonged bleeding time.
• Deficiency of FVIII associated with hemostatic disorder or of von Willebrand
factor (VWF).
• Diagnosis: low levels of VWF and FVIII due to specific or non-specific autoan-
tibody forming immune complexes.
• Treatment for acute bleeding: desmopressin (DDAVP) and VWF/FVIII concen-
trate; when refractory, recombinant FVII.
• IVIG together with corticosteroids is effective in 70% of patients.
(Yamamoto 2007)
4.5.9 B, b, d* Antiphospholipid Syndrome (APS) (*See Table 4.1)
Different forms of primary APS:
a. Venous and arterial thrombosis

b. Recurrent spontaneous abortion
c. APS associated with other autoimmune disorders (e.g., SLE)
d. Catastrophic APS with widespread thrombotic disease (CAPS)
Pathogenesis:
–– Activation of endothelial cells, monocytes, and platelets leads to procoagula-
tion serum.
–– Presence of antiphospholipid (aPL) antibodies , also named lupus anticoagu-
lant, anticardiolipin antibodies, or beta2-glycoprotein-1.
• Diagnosis: in a. often mild thrombocytopenia, hemolytic anemia or skin altera-
tions, heart valve disease, and nephropathy.
• Treatment: the results are controversial.
a. B, d* Venous and arterial thrombosis: low-dose aspirin and low-molecular
heparin; when refractory, HD IVIG (*See Table 4.1)
b. B, c, d* Recurrent spontaneous abortion: low-dose aspirin, rituximab, eculi-
zumab; adjuvant or in patients with refractory to conventional treatment, 1 g
IVIG/kg and repetition every 2 weeks, eventually after apheresis (*See Table 4.1)
c. B, d* APS associated with other autoimmune disorders: treatment as under a
(*See Table 4.1).
d. B, c, d* CAPS: triple therapy with anticoagulants, corticosteroids, plasma
exchange, and/or HD IVIG and/or rituximab (*See Table 4.1)
• Women with systemic lupus erythematosus (SLE) and recurrent abortion may
have higher rate of regular birth after HD IVIG than with corticosteroid or
NSAIDs.
(Ata et al. 2011; Tenti et al. 2016; Asherson et al. 2003)
4.5.10 B, a, c, d* Antineutrophil Cytoplasmic Autoimmune (ANCA)

Disorders (*See Table 4.1)
• Granulomatosis with polyangiitis (Wegener), eosinophilic granulomatosis with

polyangiitis (Churg-Strauss) and microscopic polyangiitis.
• Treatment: first line with immunosuppressives (corticosteroid, cyclophospha-
mide, methotrexate, azathioprine, or monoclonals (rituximab, infliximab)).
• HD IVIG as an adjuvant treatment in patients with refractory or relapsing ANCA
disorders demonstrated beneficial response.
(Jayne et al. 1993; Levy et al. 1999; Crickx et al. 2016)

42 P. Imbach
4.6 Vascular Diseases/Rheumatology
4.6.1 A, a* Kawasaki Syndrome (KS) (*See Table 4.1)
• Acute infectious or postinfectious, vascular/endothelial condition, high fever,

and with high levels of proinflammatory serum cytokines, leading to coronary
aneurysm in about 25% of children in the absence of treatment.
• Incidence is variable worldwide; in North America circa 25:100,000 children
<5 years of age per year, highest in Asia.
• Conjunctivitis, skin rash, strawberry tongue, erythema of oral and pharyngeal
mucosa, and palmar and plantar erythema.
• One dose of 2 g IVIG/kg within the first 10 days together with aspirin (<80 mg/
kg b.w.) reduces the frequency of coronary artery anomalies significantly (to
under 10%). The addition of 2 mg/kg b.w. per day corticosteroids further reduced
the complication rate (under 5%).
• In patients with persistent or recurrent fever, the risk of developing coronary abnor-
malities remains. Repetitions of the combined treatment (see above or, instead of
corticosteroids, use infliximab) or HD methylprednisolone may be indicated.
4.6.2 B, a* Henoch-Schönlein Purpura (*See Table 4.1)
• Vasculitis with purpura of the skin, sometimes with gastrointestinal, renal, and
rarely cerebral hemorrhage.
• HD IVIG is effective in patients with bleeding.
4.6.3 B, b, d, C* Juvenile Idiopathic Arthritis (JIA) (*See Table 4.1)
• JIA is a clinically heterogeneous, common chronic rheumatic disease (see below).

• Children with no sufficient response to classic treatment (nonsteroidal anti-
inflammatory drugs (NSAIDs), glucocorticosteroids, methotrexate) may be con-
sidered for treatment with HD IVIG.
• IVIG may serve as a corticosteroid-sparing option, when adverse effects are
disturbing.
• Monoclonal antibody treatment is a new line: etanercept, infliximab, adalim-
umab, tocilizumab, rituximab.
4.6.4 B
, a, b, c, d* Systemic Juvenile Chronic Arthritis (Still’s
Syndrome) (*See Table 4.1)
• Vasculitis with fever, skin lesions, and arthritis.

• In severe disorders associated with HLA-DR4.
• Laboratory: CRP high, hemoglobin low, and ANA not increased.
• HD IVIG is effective in 50%, but often transient—spontaneous recovery possible/

remission; in patients with severe, long-term disease, IVIG leads to more adequate
results than immunosuppressive treatments.
4.6.5 B, a* Felty Syndrome (*See Table 4.1)
• Variant of rheumatoid arthritis.

• Systemic inflammatory process with splenomegaly, neutropenia, occasionally
lymphadenopathy and hepatomegaly.
• HD IVIG is effective in individual patient.
(Breedveld et al. 1985)
4.6.6 B, a, e* Macrophage Activation Syndrome (*See Table 4.1)
• Severe, life-threatening variant of JLA, of SLE, of postviral infection, or after

medication or toxin exposure.
• HD IVIG may be effective, especially in early disease states.
(Stephan et al. 2001; Boom et al. 2015)
4.6.7 B, a, d* Polyarteritis Nodosa (PAN) (*See Table 4.1)
• Vasculitis of the medium and large arterioles (not capillaries as in SLE).

• Treatment: first line with immunosuppressives (corticosteroid, cyclophospha-
mide, or monoclonals (rituximab, infliximab)).
• HD IVIG demonstrated responses in reports.
(Asano et al. 2006; Breda et al. 2016).
4.6.8 B
, a, b, c, d, f * Systemic Lupus Erythematosus (SLE)
(*See Table 4.1)
• SLE is a systemic autoimmune connective tissue disease with various presenta-

tions and diverse clinical courses and prognosis.
• Standardization of treatment is difficult, and controlled studies with homoge-
neous cohort groups are not feasible.
• Different expert groups published recommendations (EULAR, ERA-EDTA, ACR).
• First-line therapeutics are hydroxychloroquine, corticosteroids, belimumab, and
aspirin.
• In patients with severe disease: cyclophosphamide, mycophenolate mofetil, and
azathioprine.
44 P. Imbach
• HD IVIG or rituximab has been effective in individual, organ-specific disorders

such as SLE-associated nephritis, myocarditis, polyradiculopathy, bone marrow
suppression, and multi-organ disease.
• HD IVIG treatment of general SLE resulted in transient improvement of 65% of
patients, in some responding patients after 6 monthly courses only.
• HD IVIG mainly remains an adjuvant treatment, a steroid sparing, and an option
for patients who are refractory to conventional treatments or in patients with
contraindication for conventional treatment.
• HD IVIG may cause prothrombotic effects in SLE, especially in SLE with neu-
rologic involvement.
(Sakthiswary and D’Cruz 2014)
4.6.9 D* Carditis in Rheumatic Fever (*See Table 4.1)
• IVIG did not show effects in prevention of cardiac sequelae of acute rheumatic
fever.
(Voss et al. 2001)
4.7 Infection-Related Diseases
4.7.1 A
, c, C * Secondary Infections in HIV Infection of Children
with B- and/or T-Cell Deficiencies Despite Taking Highly
Active Antiretroviral Treatment (HAART) or Not Taking
HAART (*See Table 4.1)
• IVIG as adjuvant treatment was effective for reducing the rates of lethality, see
also Chap. 3.12.3.
(Deener et al. 2008)
4.7.2 B
, c* Bacterial Sepsis, Septic Shock, and Streptococcal
Toxic Shock (*See Table 4.1)
• IVIG as adjuvant treatment was effective for reducing the rates of lethality; how-
ever, the result is controversial, especially in:
–– Neonatal sepsis, suspected bacterial or fungal infection in neonates
–– Group B streptococcal disease in newborn
–– Invasive streptococcal syndrome
–– Postoperative sepsis
–– Trauma-associated sepsis
• Streptococcal toxic shock: evaluations from large controlled IVIG studies of

streptococcal shock as an adjuvant treatment for children and adults (exception:
newborns; see below) demonstrated divergent results with respect to lethality.
• IVIG preventive treatment of sepsis of neonates is no longer recommended.
(Alejandria et al. 2002; Crowley and Gropper 2016; Di Rosa et al. 2014; Sallam
et al. 2016)
4.7.3 B
, a, b* Pediatric Autoimmune Neuropsychiatric Disorder
Associated with Streptococcal Infection (PANDAS)
(*See Table 4.1)
• Streptococcal infections may lead to behaviors of obsessive-compulsive tic

disturbances.
• IVIG was successful in a case-controlled study; there is an ongoing study.
(Williams et al. 2016)
4.7.4 B
, c, f * Parvovirus B19-Associated Chronic Fatigue
(*See Table 4.1)
• The prevalence of B19 infections is high in the general population and in patients
with autoimmune disorders.
• IVIG or SCIG may decrease viremia and proinflammatory cytokine levels and
improve chronic fatigue syndrome.
(Kerr et al. 2003; McGhee et al. 2005)
4.8 Neurology (See Also Chap. 8)
Overview: Ref. Lünemann et al. (2015)
4.8.1 A, a* Guillain-Barré Syndrome (GBS) (*See Table 4.1)
• Pathogenesis: mostly postinfectious (systemic CMV infection, gastroenteritis by

Campylobacter jejuni) polyneuroradiculitis with antibodies cross-reacting with
ganglioside antigen activating complement and causing demyelination of periph-
eral nerves.
• Clinical diagnosis: ascending acute progressive motor weakness, often starting
in the lower extremities, autonomic nerve dysfunction (diaphragmatic muscles
with lung dysfunction resulting in mechanical ventilation), and bulbar and facial
muscle dysfunction, the latter with inability to walk.
46 P. Imbach
• GBS variants are the acute axonal motor or motor sensory form (Miller Fisher
syndrome) and the acute dysautonomia form.
• Treatment: IVIG (0.4 g/kg b.w./day × 3–5) and/or corticosteroids or plasma
exchange depending on the grade of severity of the progression.
(Hughes et al. 2007, 2014; van Koningsveld et al. 2004; van Doorn et al. 2010)
4.8.2 A
, a* Chronic Inflammatory Demyelinating
Polyneuropathy (CIDP) (*See Table 4.1)
• Most common autoimmune peripheral neuropathy.

• Pathogenesis: immunological progressive destruction of the myelin sheaths of
peripheral nerves.
• Any target antigen is detectable, electrophysiological signs of demyelination
with conduction block.
• Clinical diagnosis: slow onset of symmetrical weakness of muscles with a reflexia
and sensory loss, progressive over months.
• Treatment: IVIG or SCIG 16 (2 g/kg within 5 days) with repetitions (1 g/kg)
every 3 weeks improves disabilities within 2–6 weeks and increases quality of
life; response rate of 60% is not only transient but also long-term improvement
with strong impacts of quality of life.
• Alternative treatments are plasma exchange and/or high-dose
methylprednisolone.
(Dalakas et al. 2011; Dalakas et al. 2015; Berger et al. 2008; Eftimov et al. 2009;
Hughes et al. 2008)
4.8.3 A, a* Multifocal Motor Neuropathy (MMN) (*See Table 4.1)
• Pathogenesis: chronic progressive inflammatory disease asymmetrically affect-

ing the motor nerves.
• The weaknesses mainly occur at radial, ulnar, and common peroneal muscle
nerves.
• Treatment: IVIG or SCIG (0.4 g/kg/day × 5) with 60–80% improvements of
muscle strength and scores.
• An alternative is subcutaneous IgG (SCIG) administration, which had the same
efficacy as IVIG in a small treatment study.
• IV/SCIG repetitions with 1–2 g/kg, if unsatisfactory or unsustained
response.
• In contrast to GBS and CIDP, corticosteroids and plasma exchange are not effec-
tive in MMN.
(Hahn et al. 2013; Leger et al. 2001; Harbo et al. 2010)
4.8.4 B, a, b, d, f * Myasthenia Gravis (*See Table 4.1)
• Pathogenesis: neuromuscular junction deficiency of stimulation of muscles result-

ing in muscle weakness including occasionally bulbar or respiratory failure.
• Associated with antibodies against the acetylcholine receptor or against muscle-
specific kinase.
• Two patterns of clinical manifestation: early onset without thymoma, mostly in
women, and late onset with thymoma.
• Clinical symptoms: asymmetric ptosis, diplopia; less common, oropharyngeal
and limb weakness.
• Treatment: individual approach.
• First line: cholinesterase inhibitors.
• Among corticosteroids and plasma exchange, HD IVIG had equal or higher
response rates of decrease in acetylcholine receptor antibodies and clinical effi-
cacies 6 weeks after IVIG administration.
• The tolerance of IVIG is higher, adverse effects are lower, and IVIG has steroid-
sparing effects.
• Efficacy of IVIG has been demonstrated in juvenile myasthenia and myasthenic
exacerbations.
• IVIG is indicated in preparation of thymectomy.
(Alabdali et al. 2014; Wolfe et al. 2002; Gajdos et al. 2012)
4.8.5 B
, a* Lambert-Eaton Myasthenic Syndrome (LEMS)
(*See Table 4.1)
• Rare disease of neuromuscular, presynaptic signal transmission in patients with

autoantibodies against the presynaptic calcium channel at the motor end plates.
• Also observed as paraneoplastic conditions, mainly in patients with small-cell
lung cancer.
• Symptoms: proximal muscle weakness, with low tendon reflexes.
• HD IVIG showed transient efficacies (clinical, antibody level, and in nerve stim-
ulation) within 2–4 weeks. It is an alternative therapy for immunosuppressive
treatments with less adverse effects (see above).
(Skeie et al. 2010)
4.8.6 B, a* Dermatomyositis (*See Table 4.1)
Dermatomyositis is described in Sect. 4.9.1.
4.8.7 B, a, d, f * Multiple Sclerosis (MS) (*See Table 4.1)
• Clinical manifestation (wide variety): optic neuritis, dizziness, bladder dysfunc-

tion, sensory impairment.
48 P. Imbach
• Treatment is complex with individualized approach; corticosteroids and/or beta-

interferon still are the first-line treatment.
• In some early studies, long-term administration of 0.2–2 g IVIG/kg b.w. monthly
reduced the relapse rate, the disability scores, the rate of progress/deterioration,
and the number and volume of detected lesions by MRI; later on, controlled stud-
ies showed any significant improvement within IVIG in remitting-relapsing and
progressive MS.
• IVIG is recommended as a second-line treatment along with beta-interferon and/
or corticosteroids.
• IVIG treatment in patients with early stages of MS and pregnant women with MS
showed less relapses.
• In patients with severe progressive or refractory MS, rituximab may be
considered.
• New immunomodulators such as monoclonal antibodies (alemtuzumab, natali-
zumab) and drugs will define the role of IVIG
(Sorensen et al. 2002; Fazekas et al. 2008).
4.8.8 B, a, d, f * Neuromyelitis Optica (*See Table 4.1)
• Central nervous inflammatory, demyelinating disease with autoantibodies against

components of astrocytes (aquaporin-4).
• IVIG as first- or second-line long-term treatment (similar as in multiple sclero-
sis) decreases the rates of relapses and the progression of disabilities.
(Magraner et al. 2013)
4.8.9 B
, a, d, e, f * Intractable Epilepsy in Children and Refractory
Pediatric Epilepsy (*See Table 4.1)
• Lennox-Gastaut syndrome, West syndrome, and myoclonic encephalopathy are

the classic examples in children with imbalanced immune responses.
• HD IVIG reduced the frequency of seizures.
• Children with refractory epilepsy and low quality of life profit from a treatment
with IVIG.
(Billiau et al. 2007)
4.8.10 B, a, d, f * Rasmussen Syndrome (*See Table 4.1)
• Characterized by focal seizures, progressive neurological and intellectual dete-

rioration, chronic encephalitis, and hemispheric atrophy.
• HD IVIG reduces seizures, but not the progression of the disease, compared with
HD corticosteroids in children and adults.
(Hart et al. 1994)
4.8.11 B, a, c, e, f * Neuroimmunological Conditions, Disorders

with Autoantibodies Against Nervous Tissues/Receptors/
Other Antigens (*See Table 4.1)
• Examples are acute disseminated encephalomyelitis, autoimmune encephalitis,

demyelinating brain stem encephalitis, subacute rhombencephalitis optica,
opsoclonus-myoclonus syndrome.
• Besides immunosuppressive treatment in patients with one of the treatment men-
tioned above, rare diseases associated with autoantibodies IVIG may justify a
trial for severe or treatment refractory, for individual patients.
• Even small controlled studies are not feasible in those conditions.
(Nosadini et al. 2015)
4.8.12 C D* Polyneuropathy Associated with Monoclonal IgM

Gammapathy (*See Table 4.1)
• Inclusion myopathy, idiopathic neuropathies, brachial plexopathy, diabetic amy-

otrophy, adrenoleukodystrophy, autism without primary immunodeficiency.
• These disorders didn’t show evidence of improvement after IVIG administration.
4.8.13 B, a, f * Stiff Person Syndrome (SPS) (*See Table 4.1)
• Stiff person syndrome is characterized by muscle rigidity and spasms and with
high anti-glutamic acid decarboxylase antibodies.
• Patients may benefit from IVIG administration resulting in reduced stiffness and
daily activities as well as improved quality of life.
(Dalakas et al. 2005)
4.8.14 B a (A c)* Chronic Fatigue Syndrome Associated

with Immune Dysfunctions (*See Table 4.1)
• Case and anecdotal reports of patients with disorders associated with immune
dysfunctions (e.g., ITP) may profit from IVIG administration.
(Twisk et al. 2014)

50 P. Imbach
4.8.15 C D* Alzheimer’s Disease (*See Table 4.1)
• The studies on IVIG in Alzheimer’s disease are controversial.

• The clinical disease assessment Scale-Cognition demonstrated reversal of dis-
ease progression, decreased beta-amyloid peptide levels in the cerebrospinal
fluid, and improved cognitive function.
• Another controlled study did not support the efficiency of IVIG.
(Dodel et al. 2013; Relkin et al. 2017)
4.9 Dermatology
Overview: Enk et al. 2017
4.9.1 A, b, B, b* Dermatomyositis (*See Table 4.1)
• Rare, heterogenous conditions of mostly acquired inflammatory muscle diseases

classified as:
–– Dermatomyositis (DM)
–– Polymyositis (PM)
–– Necrotizing myositis (NM)
–– Inclusion body myositis (IBM) (category C)
–– Overlapping disorder with SLE, Sjögren’s syndrome, rheumatic arthritis
(RA), systematic vasculitis
• Incidence: DM 2–9/1 Mio. population/year, IBM 4.5–9.5/1 Mio. population/year
increasing to over 35/1 Mio. in over 50-year-old persons.
• Pathogenesis: T-cell-mediated autoimmunity and antibody-mediated effector
mechanisms with endomysial capillary destruction.
• Symptoms:
–– Cutaneous: rash on the face and extremities (DM), periorbital edema, dystro-
phic nails, alterations of hairy scalp.
–– Cutaneous manifestations are variable, also present without muscle involve-
ment = amyopathic DM (3–20%).
–– Muscles: symmetrical proximal muscle weakness; also pharyngeal, respira-
tory, cardiac, and neck muscles may be affected (pulmonary function test!).
–– Thirty percent of adults with DM are associated with malignancy.
• Laboratory/radiologic diagnosis: high creatine kinase (CK), CRP, and muscle
aldolase and low serum complement (by consumption), EMG, muscle biopsy,
and MRI.
• Different myositis-specific antibodies can be detected (not yet part of routine
diagnosis).
• Treatment:
–– First line: Corticosteroids.
–– Adjuvant: azathioprine in adults, methotrexate in children.
–– In patients with fulminant progressive disease, with refractory/insufficient

response, HD IVIG courses every 1–2 months.
–– In responders to IVIG, maintenance therapy for 1–3 years.
–– In some patients antimalaria agents (hydrochloroquine) in combination with
the conventional regimen may be indicated.
–– HD IVIG led to significant improvement in muscle strength within 3 months,
occasionally in combination with rituximab, and with corticosteroid-sparing
effects, especially in children.
–– HD IVIG is used in patients who are unresponsive to conventional treatment
or to avoid side effects.
–– In the future, subcutaneous IG (SCIG) with smaller doses/week is feasible,
effective, and safe.
–– Cutaneous manifestation: in addition to systematic treatment, topical cortico-
steroids and ultraviolet light.
–– Long-term management with physical exercise.
• Prognosis: remission in 40%, partial remission in 43%, and exacerbation in 17%.
• In patient with IBM, IVIG and corticosteroids are ineffective.
(Dalakas 1995, 2015; Dalakas et al. 1993; Sunderkötter et al. 2016; Cherin et al. 2016)
4.9.2 A
, a, B, f (C)* Dermatologic Mucocutaneous Autoimmune
Disorders (*See Table 4.1)
• Pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigoid, and

epidermolysis bullosa (blistering skin diseases).
• Mucocutaneous bullous conditions in which intraepidermal or subepidermal lay-
ers of the skin are separated due to antibodies against intracellular adhesion mol-
ecules, such as desmoglein.
• Chronic, relapsing-remitting autoantibody-mediated skin lesions with life-
threatening complications.
• Treatment: IVIG as a parallel adjuvant treatment of immunosuppressive thera-
pies (corticosteroid, azathioprine, mycophenolate mofetil, rituximab).
• Dosage and duration of IVIG administration: 2 g/kg b.w. in 2–5 doses initially
and then monthly for a period of 6 months. If efficacious the interval of adminis-
tration may be increased to 5–6 weeks. In parallel, the dosage of concomitant
immunosuppressants (corticosteroid, cyclophosphamide, azathioprine, and
others) can often be reduced.
• IVIG as a second line treatment in patients with relapsing disease or refractory to
other first-line treatments.
• Regular intervals without IVIG should be attempted. And after 6 months wash-
out period of responders should be considered.
• IVIG as first-line treatment with low or any immunosuppressants should be con-
sidered during pregnancy, in infants, and adolescents, if immunosuppressive
treatment is contraindicated or in disorders with immunodeficiencies.
(Enk et al. 2017)

52 P. Imbach
4.9.3 B
, a, c, d, e* Stevens-Johnson Syndrome and Toxic
Epidermolysis (*See Table 4.1)
• Severe, occasionally fatal disorder associated with Fas (CD95) apoptosis.

• Early administration of HD IVIG (together with corticosteroids) is effective with
no further progression and reduction of lethality.
(Viard et al. 1998; Huang et al. 2012)
4.9.4 B, a, e, f * Photodermatosis/Solar Urticaria (*See Table 4.1)
• This rare skin disorder showed recovery with IVIG after 3–6 courses of 0.4 g/kg b.w.
(Adamski et al. 2011)
4.9.5 B
, a, d* Scleromyxedema and Variants: Systemic Sclerosis/
Scleroderma with Skin Involvement, Linear Scleroderma,
Morphea, Mixed Connective Tissue Disease, Sjögren’s
Syndrome (*See Table 4.1)
• Scleromyxedema is a severe condition characterized by fibroblast proliferation

and mucin deposition in the skin and organs, often associated with monoclonal
gammopathy.
• Symptoms: thickening of the skin with debilitating alterations.
• Treatment: often refractory to immunosuppressive treatment.
• HD IVIG is often the treatment of choice in severe, refractory situations, over a
period of months in responding patients.
(Bidier et al. 2012; Jolles and Hughes 2006)
4.9.6 B
, a* Chronic Urticaria and Delayed Pressure Urticaria
(*See Table 4.1)
• One third of patients with chronic urticaria have a related autoimmune process.
• HD IVIG may play a role in the aforementioned patients.
(Mitzel-Kaoukhov et al. 2010)

4.9.7 B, b, c, d, f * Atopic Dermatitis (AD) (*See Table 4.1)
• In children and infants with severe, refractory to first-line treatment of AD, HD

IVIG (2 g/kg b.w./4 weeks) for 3–6 months showed significant clinical improve-
ment, decreases of eosinophils and serum IgE after 3 months, and remissions at
6 months.
• Children with severe, refractory AD and recurrent Staphylococcus aureus or
herpes simplex superinfection had partial or complete remissions within

6–>12 months after HD IVIG treatment (dosage similar as mentioned above).
• Adults with severe atopic dermatitis show less dramatic results as reported in
children (see above).
(Jee et al. 2011)
4.10 Organ-Specific Diseases
4.10.1 A, c* Cystic Fibrosis (*See Table 4.1)
• No evidence of IVIG indication in controlled studies of patient with cystic fibro-

sis without immunodeficiencies
(Winnie et al. 1989; Balfour-Lynn et al. 2004; Smyth 2004)
4.10.2 A, c* Asthma (*See Table 4.1)
• IVIG may be indicated as an adjuvant replacement treatment in immunodeficient

patients with asthma and with chronic inflammations and respiratory symptoms.
(Schwartz and Berger 2002)
4.10.3 A, c* CMV Pneumonitis (*See Table 4.1)
• IVIG or anti-CMV IVIG in combination with ganciclovir in relation to trans-

plantation prolongs the survival of immunodeficient patients.
(Ljungman et al. 1992)

54 P. Imbach
4.10.4 B, a, b, d, f (A c)* Solid Organ Transplantation (*See Table 4.1)
• Secondary immunodeficiency due to rituximab and other immunosuppressive

regimens or due to antibody-mediated rejection of transplants are the rationale to
administer IVIG.
(Burton et al. 2015)
4.10.5 B, a, c* Autoimmune Ophthalmopathy (Graves) (*See Table 4.1)
• Associated with hyperthyroiditis involving orbital tissues and autoantibodies.

• Main symptom: proptosis.
• Treatment: HD IVIG (6 courses, interval 3 weeks) versus corticosteroids demon-
strated equal effects, but less adverse effects in the IVIG group.
• Recently, rituximab showed good responses in patients with severe disease.
(Kahaly et al. 1996)
4.10.6 B, a, d, e, f * Autoimmune Uveitis (*See Table 4.1)
• Inflammation of the vascular layers of the eye

• Ranges from visual impairment to blindness
• Treatment:
–– First line: corticosteroids or monoclonal.
–– In patients with refractory disease or with birdshot retinochorioidopathy,
IVIG was successful in 50% of patients with visual improvement.
(Prete et al. 2016)
4.10.7 B, a, b, d, e, f * Autoimmune Liver Disease (*See Table 4.1)
• Pathology: chronic active autoimmune hepatitis with high serum liver enzymes,
immune complexes, and periportal mononuclear infiltrates.
• HD IVIG improves the pathological findings with less adverse effects than with
immunosuppressives (corticosteroids, rituximab).
(Vierling and Flores 2002)
4.10.8 B, d* Inflammatory Bowel Disease (*See Table 4.1)
• Inflammatory manifestation of the whole gastrointestinal tract of Crohn disease

or of ulcerative colitis (lower colon).
• IVIG may be effective in immunosuppressiva refractory patients.
(Merkley et al. 2015)
4.10.9 B, a, d, e, f * Acute Myocarditis (*See Table 4.1)
• HD IVIG improved cardiac function and decreased viral load in parvovirus B19-
associated cardiomyopathy.
(Dennert et al. 2010)
4.10.10 B, a, c* IgA Nephropathy (*See Table 4.1)
• A report of six patients demonstrated stabilization and delayed progression of

loss of renal function after administration of IVIG.
(Rasche et al. 2006)
4.10.11 B, a, b* Autoimmune Diabetes (*See Table 4.1)
A subgroup of patients with antibodies against islet cells responded to IVIG treat-
ment with higher rates and longer duration of remission.
(Heinze 1996)
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Anti-D: A Type of IVIg
5
Ramsha Khan and Alan H. Lazarus
5.1 Introduction
5.1.1 Anti-D as an IVIg
Rh immune globulin (anti-D) is a polyclonal IgG antibody directed against the

Rhesus D (RhD) factor present on red blood cells (RBCs). It is a type of intravenous
immunoglobulin (IVIg, which contains antibodies of the IgG isotype) isolated from
the pooled plasma donated by two types of alloimmunized donors:
1. Rhesus D-negative (RhD−) male donors who have been intentionally immu-
nized with RhD+ RBCs (Crow and Lazarus 2008; Semple 2010)
2. RhD− female donors who have been naturally immunized by Pregnancy with an
RhD+ fetus (Semple 2010)
It is worth noting that although anti-D may be the desired antibody present in the
preparation isolated from plasma, a variety of other antibodies will invariably also
be included in the final solution, as will be discussed later (Crow and Lazarus 2008).
R. Khan, HBSc
Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of
Toronto, Toronto, ON, Canada
Keenan Research Centre of the Li Ka Shing Knowledge Institute at St. Michael’s Hospital,
Toronto, ON, Canada
A.H. Lazarus, PhD (*)
Toronto, ON, Canada
Centre for Innovation at The Canadian Blood Services, Ottawa, ON, Canada
Departments of Medicine and Laboratory Medicine & Pathobiology, Faculty of Medicine,
University of Toronto, Toronto, ON, Canada

62 R. Khan and A.H. Lazarus
Being a donor-derived product, anti-D is available in limited quantities and car-

ries a theoretical risk of transferring emerging pathogens and infectious diseases
(Brinc and Lazarus 2009). Ethical concerns also stem from intentionally immuniz-
ing a large number of RhD− men (as only 15–20% are responders) with the RhD+
cells for the sole purpose of manufacturing anti-D.
5.1.2 Clinical Uses of Anti-D
Currently anti-D is used clinically to prevent hemolytic disease of the fetus and
newborn (HDFN) and to treat immune thrombocytopenia (ITP) (Lazarus 2013;
Semple 2010). It may also be used to clear RhD+ RBCs from the bloodstream when
they have accidentally been transfused to RhD− individuals.
Hemolytic disease of the fetus and newborn (HDFN) occurs when alloimmuni-
zation causes the maternal immune system to destroy fetal RBCs expressing differ-
ent, and thus, foreign, antigens when compared to the mother’s RBCs (Hendrickson
and Delaney 2016). The most predominant antigen causing HDFN is the D antigen
(others include the Kell antigen and the Duffy antigen). RhD− mothers can be
exposed to RhD+ RBCs, either through blood transfusions or from previous preg-
nancies with an RhD+ fetus (Hendrickson and Delaney 2016). Once the mother has
been sensitized, subsequent pregnancies with an RhD+ fetus may be at risk of
HDFN as the anti-D produced by the maternal immune system can potentially cross
the placenta and destroy the fetal RBCs (Urbaniak and Greiss 2000). Currently, only
prophylactic anti-D injections are able to prevent HDFN from occurring, and in
many countries, the priority is to conserve it for HDFN prophylaxis instead of utiliz-
ing it to ameliorate ITP as discussed below (Lazarus 2013).
ITP is an autoimmune disorder characterized by low platelet levels primarily due
to clearance mediated by platelet-reactive autoantibodies (Lazarus 2013). These
antibodies are predominantly of the IgG1 and IgG3 isotypes although IgM and IgA
subtypes may also be present (Semple 2010). In severe cases, ITP patients may
experience lower energy levels and are at an increased risk of life-threatening bleed-
ing. Some of the therapies for ITP include steroids, IVIg, and anti-D. First shown by
Imbach et al. in 1981, high doses of IVIg are able to significantly raise platelet lev-
els in ITP patients (Imbach et al. 1981). However the mechanism(s) through which
IVIg is able to ameliorate ITP remains unclear even today. Anti-D is considered to
be a first-line treatment for ITP and is able to ameliorate ITP at approximately 5-log
fold lower dose than IVIg—50–75 ug/kg of anti-D is generally as effective as 1–2 g/
kg of IVIg administered to ITP patients (Eghbali et al. 2016). Moreover, compared
to other monoclonal antibodies that are used to modulate autoimmune diseases like
ITP (such as anti-CD20; Rituximab), anti-D is administered at an approximately
2-log fold lower dose for a similar therapeutic response (Rosman et al. 2013; Nair
and Jacob 2016). Nevertheless, like IVIg, the mechanism through which anti-D is
able to ameliorate ITP also remains unclear. Research has proposed a few potential
mechanisms which will be discussed later. If the first-line treatments are
5 Anti-D: A Type of IVIg 63
unsuccessful, other therapies involving anti-CD20, thrombopoietin, splenectomy

and others may be considered (Semple 2010).
5.1.3 Black Box Warning on Anti-D Usage by the FDA
In 1995, the food and drug administration (FDA) granted approval for utilizing anti-
D as a therapeutic for ITP patients. However in 2010 the FDA issued a black box
warning for all anti-D products used in ITP. This mandated warning, to be printed
on the package insert, is used to signify an increased risk of intravascular hemolysis
associated with anti-D usage. It cautions about the increased risk of developing
anemia, acute renal insufficiency, renal failure, disseminated intravascular coagula-
tion (DIC), multisystem organ failure including acute respiratory distress syndrome
(ARDS), and death. The warning also provides physicians with guidelines on how
to proceed in the care of patients that had been injected with anti-D (FDA, 2009).
In response to the FDA-mandated warning, a working group was assembled at
the instigation of Cangene bioPharma to evaluate the safety of anti-D. This panel of
researchers, consisting of physicians and some scientists affiliated with the biotech-
nology industry, evaluated the safety data provided by Cangene and concluded that
the safety profile of anti-D in ITP is not significantly different than that of other
therapies, such as IVIg (Despotovic et al. 2012). The researchers also indicated that
the adverse side effects can be prevented through careful selection of patients (e.g.,
excluding patients with predisposing conditions that may lead to an increased risk
of DIC and ARDS) (Despotovic et al. 2012).
5.2 Potential Mechanisms of Action of Anti-D in ITP
The underlying mechanism(s) through which anti-D acts to ameliorate ITP remains
unclear. Previous and ongoing work has allowed researchers to propose a number of
mechanisms that may be at play—these include mononuclear phagocytic system
(MPS) blockade, cytokine modulation, and the presence of anti-idiotype antibodies
(Semple 2010; Crow and Lazarus 2008).
Much of the research related to mechanisms of anti-RBC antibodies in ITP has
used mice as a model system. In the murine model of passive ITP, mice are injected
with an antiplatelet antibody, such as MWReg30 which binds the glycoprotein IIb/
IIIa (also known as αIIbβ3 or CD41/CD61) complex present on the surface of plate-
lets and leads to the development of thrombocytopenia. However the D antigen on
murine RBC is not immunogenic, and thus, other RBC antigens (and the corre-
sponding antibodies) are used as surrogates to mimic the anti-D effect. One of the
most common antibodies used in lieu of anti-D is TER-119. TER-119 binds the
glycophorin-A (GPA)-associated protein on murine RBCs and has been shown pre-
viously to work successfully in murine models of ITP (Crow and Lazarus 2008;
Deng and Balthasar 2007).
5.2.1 Mononuclear Phagocytic System (MPS) Blockade
The mononuclear phagocytic system (MPS) is a part of the immune system that
includes macrophages. Macrophages are a major phagocytic cell population in the
immune system and are found in most body tissues (Hume 2006). Macrophages
express Fc receptors (FcRs) on their cell surface that bind the Fc portion of an anti-
body. These Fc receptors can be classified based on the type of antibody they bind—
the most important ones for mediating phagocytosis of opsonized particles (such as
RBCs and platelets) are the Fc-gamma receptors (FcγR) that bind IgG antibodies
(Semple 2010). The human FcγR family includes several activating members (such
as FcγRI, FcγRIIA, and FcγRIII) and an inhibitory member (FcγRIIB) that differ in
their affinity to bind different molecular structures of IgG and in their ability to
potentiate or inhibit immune responses (Bruhns and Jönsson 2015).
Salama et al. (1984) were the first to demonstrate the efficacy of anti-D in ame-
liorating ITP. Although the mechanism was not elucidated, they had postulated that
anti-D was exerting its therapeutic effects through MPS blockade. The MPS block-
age theory hypothesizes that since the RBCs are present in a much larger quantity
than platelets, anti-D-opsonized RBCs competitively inhibit platelet phagocytosis
by binding free FcγRs on macrophages, (Fig. 5.1) particularly those that are present
in the spleen which is a primary site of MPS activity (Salama et al. 1983, 1984;
Crow and Lazarus 2008). Over the years, MPS blockade was indirectly supported
by various studies as the mechanism of action of anti-D in ITP (Becker et al. 1986;
Bussel et al. 1991; Ware and Zimmerman 1998; Ambriz-Fernández et al. 2002).
Evidence supporting this theory includes the therapeutic inefficacy of anti-D in ITP
patients that are RhD− (Boughton 1991; Oksenhendler et al. 1988). However, of
these RhD− patients, those who were positive for the Rh c antigen (on RBCs) did
respond to anti-c therapy, and an increase in platelet counts was observed
(Oksenhendler et al. 1988). This suggested that anti-RBC antibody (against the c, D,
or presumably any other Rh antigen present on the surface of RBCs) sensitized red
blood cells and blocked MPS consequently preventing platelet phagocytosis. More
evidence supporting MPS blockade as the mechanism of action of anti-D stems
from the observation that ITP patients who have previously had a splenectomy do
not respond well to anti-D therapy (Bussel et al. 1991); however it has also been
found that two splenectomized ITP patients did in fact respond to anti-D therapy
(Ramadan and El-Agnaf 2005). Thus, more work is required to clearly determine
whether or not anti-D functions (at least partially) through MPS blockade and how
the presence and absence of a spleen affects that function.
5.2.2 FcγRs
As indicated previously, macrophages can bind antibodies through FcγRs expressed

on the cell surface. These receptors may be involved in activating phagocytosis,
such as FcγRIIIA, or inhibiting it, such as FcγRIIB. To our knowledge, no reports
have actually observed macrophage FcγR-mediated phagocytosis of
anti-D-opsonized RBCs. However much work has been done regarding the role of
FcγRs in murine models of ITP using surrogate antibodies (such as TER-119).
It was recently shown that the Fc portion of the anti-RBC antibody TER-119 is
required for ITP amelioration in a murine model (Yu et al. 2015). The results show
that although the F(ab’)2 region of TER-119 alone is able to bind to the RBCs, it is
unable to mediate phagocytosis. The F(ab’)2 region of TER-119 was unable to
increase platelet levels in an ITP model or cause significant anemia. The same
results were observed for TER-119 with a deglycosylated Fc region (Yu et al. 2015)
demonstrating that both the Fc domain of TER-119 and Fc glycosylation are
required for ITP amelioration and for inducing anemia. On the other hand, an earlier
study done with TER-119 but with a murine model of autoimmune hemolytic ane-
mia (AHA) lacking FcRr chains (the signaling component of activating FcrRs in
mice) chains had concluded that FcγR-mediated mechanisms are not required by
TER-119 to cause anemia (Chen et al. 2014). These seemingly differing results
indicate that TER-119 may be inducing anemia through multiple different mecha-
nisms in ITP and AHA and that in the ITP model, the native glycosylated Fc domains
of the antibody are essential for disease amelioration.
The above results indicated that at least in murine passive ITP, FcγR binding is
required for amelioration. Earlier work had attempted to determine which FcγRs
might be involved. In 2005 Song et al. established a correlational relationship
between anti-RBC antibody-mediated increase in platelet levels and the downregu-
lation of FcγRIIIA on splenic macrophages in a murine model of ITP (Song et al.
2005). FcγRIIIA is an activating receptor found on the surface of macrophages, and
based on the results of this study, it is likely that these anti-RBC antibodies modu-
late, inactivate, or neutralize FcγRIIIA or the FcγRIIIA pathway (Lazarus 2013).
This, in turn, suggests that inhibition of MPS is, at the very least, a partial contribu-
tor to the mechanism of action of the anti-RBC antibodies used.
It was also shown that anti-RBC antibodies are able to ameliorate ITP in mice
lacking FcγRIIB (Song et al. 2005). FcγRIIB is considered to be an inhibitory
receptor present on macrophages, and when activated, it has been suggested to
downregulate the phagocytic activity of the macrophage. Later research per-
formed by Yu et al. in 2015 again found that TER-119 was able to ameliorate ITP
in mice lacking inhibitory this receptor (FcγRIIb−/−) indicating that these recep-
tors are not required by the anti-RBC antibodies used to perform their function in
ITP (Yu et al. 2015).
The research thus far had shown anti-RBC antibodies might be dependent on
FcγR-mediated mechanisms to ameliorate ITP (e.g., by neutralizing FcγRIIIA func-
tion) and that the inhibitory receptor FcγRIIB was probably not an essential compo-
nent of this mechanism. Building on this, further work showed that mice treated
with the Fab region of a monoclonal antibody 2.4G2, an inhibitor of both FcγRIIB
and FcγRIIIA, and with antiplatelet antibody (to induce ITP) developed a similar
degree of anemia as the control group when treated with TER-119. This indicated
that these receptors might not be required by the TER-119 anti-RBC antibody to
cause anemia—however in vitro assays showed partial inhibition of RBC phagocy-
tosis by RAW264.7 macrophages treated with 2.4G2. Combining this with the ear-
lier results, at least two possibilities emerge:
1. That the anemia-inducing effects of an anti-RBC antibody may not be necessary

for ameliorating ITP
2. That the in vivo vs in vitro role of FcγRs related to anti-RBC-mediated effects
differs (Yu et al. 2015).
Taken together, some potential concepts advanced by the various studies include:
1. That IgG Fc domains and IgG Fc glycosylation of anti-RBC antibodies may be

required for murine ITP amelioration
2. That anti-RBC antibodies may potentially interact with multiple FcγRs to initi-
ate their ameliorative effects
3. That FcγRIIB expression is not required by anti-RBC antibodies to ameliorate
murine ITP
4. That anti-RBC antibodies may modulate or inactivate the FcγRIIIA pathway
5.2.3 Cytokine Modulation
Numerous groups have documented the ability of anti-D to induce cytokine modu-
lation. In 1994, Davenport et al. demonstrated in in vitro experiments that anti-D-
opsonized red blood cells stimulated peripheral blood mononuclear cells (PBMCs)
to secrete interleukin-1 receptor antagonist (IL-1Ra) (Davenport et al. 1994). IL-1Ra
is structurally similar to IL-1 and can bind its receptor with equal affinity but is
unable to induce signal transduction—thus IL-1Ra acts as a competitive inhibitor of
IL-1 (Dripps et al. 1991).
Semple et al. followed up on this observation with a study in children with
chronic ITP and demonstrated that along with IL-1Ra, several other pro- and anti-
inflammatory cytokines and chemokines were present at increased levels. These
included IL-6, GM-CSF, MCP-1α, TNF-α, and MCP-1 (Semple et al. 2002).
Notably, IL-1Ra levels in serum were approximately 60-fold higher than baseline
within 3 h of anti-D administration but returned to baseline by day 8 (along with the
other cytokines and chemokines that were also observed to be elevated).
The researchers further went on to show that early increases in IL-1Ra levels
were correlated with decreased platelet phagocytosis. An in vitro assay demon-
strated that IL-1Ra directly inhibited the phagocytosis of anti-D-sensitized RBC by
monocytes and granulocytes (Coopamah et al. 2003). However Crow et al. (2007)
demonstrated in a murine model that while TER-119 was able to increase IL-1Ra
levels, it was also able to ameliorate ITP in mice lacking the IL-1R receptor, the
receptor for IL-1 and IL-1Ra. This indicated that at least in mice, IL-1R may not be
essential for amelioration of ITP. This difference in the human and murine studies
performed by Semple et al. and Crow et al., respectively, may be attributed to sev-
eral reasons including the differences between species and methodologies (Semple
2010). It may also be because of differences between the antibodies used—TER-
119 is able to mimic anti-D effectively in murine models, but it is in fact a surrogate
antibody which may potentially account for some of the differences observed.
Further research is needed to help clarify the role and significance of IL-1Ra modu-
lation in anti-RBC antibody-mediated ITP amelioration.
Other groups have also supported Semple et al.’s finding of increased levels of
certain chemokines and cytokines (Malinowska et al. 2001; Branch et al. 2006;
Cooper et al. 2004). Specifically, Cooper et al. found that 2 h after the anti-D treat-
ment, there were increased levels of IL-6, IL-10, TNF-α, and MCP-1. This is in
contrast with IVIg which induced an increase only in IL-10 levels 2 h posttreatment,
suggesting that IVIg and anti-D may be exerting their therapeutic effects via differ-
ing mechanisms (Cooper et al. 2004).
Researchers have also shown that anti-D-induced cytokine changes in vitro can
lead to a rapid (within 10–30 min) but transient increase in the production of reac-
tive oxygen species, ROS, by human macrophages (Coopamah et al. 2003; Branch
et al. 2006). This production of ROS induced by anti-D administration may be acti-
vating the MPS by providing “danger” signals (Semple 2010). The significance of
these anti-RBC antibody-mediated changes in cytokine levels still remains to be
elucidated fully.
5.2.4 Anti-idiotype Antibodies
An idiotype is composed of a collection of different antigenic determinants, known

as idiotopes, in the variable region of the antibody. Anti-idiotype antibodies recog-
nize and bind these antigenic determinants on other antibodies. Anti-idiotype anti-
bodies play a role in immune system development and regulation, as proposed by
Niels Kaj Jerne in his Nobel Prize winning immune network theory [reviewed in
Semple (2010)].
Since anti-D is a polyclonal IgG enriched from donated plasma, it is expected
that this preparation will contain various anti-idiotype antibodies. Boughton et al.
showed that ITP patients that responded to anti-D treatment had a concomitant
decrease in the platelet-associated autoantibodies directed against cell surface gly-
coproteins IIb/IIIa (Boughton et al. 1994). This finding supports the fact that anti-D
contains neutralizing anti-idiotype antibodies that bind platelet autoantibodies and
thus prevents them from inducing thrombocytopenia.
On the other hand, a commercial blend of 25 different monoclonal antibodies
against the RhD antigen on human RBCs, rozrolimupab, was found to significantly
elevate platelet levels in adult human patients with ITP at high doses (Lazarus
2013). Rozrolimupab would not be expected to contain anti-idiotype antibodies,
indicating that these anti-idiotype antibodies are probably not essential for anti-D-
mediated ITP amelioration. It has also been shown that murine monoclonal anti-
RBC antibodies that mimic anti-D (and do not contain any anti-idiotype antibodies)
are able to efficaciously increase platelet levels in murine models of ITP (Song et al.
2003). The efficacy of both rozrolimupab and monoclonal murine anti-RBC anti-
bodies in ITP suggests that anti-idiotype antibodies are likely not the primary mech-
anism of action of anti-D although more research is required to confirm and assert
this conclusion.
5.3 Replacement Strategies
Monoclonal replacements for anti-D have been tested in HDFN with mixed
results—many were observed to decrease RBC immunization as desired although to
a lesser extent than polyclonal anti-D, while others actually worsened the immune
response (Kumpel 2007).
For ITP, one single monoclonal antibody was tested in seven adult patients in
the late 1990s but led to disappointing results. It was found to bind RBCs and
cause a similar degree of anemia as anti-D. However, there was little to no
increase observed in platelet counts for six patients, while the seventh patient
with mild ITP showed a transient response (Godeau et al. 1996). This study
hinted at the possibility that perhaps monoclonal anti-D therapy is not efficacious
for ITP patients. Following the publication of the results of this study, many
researchers worked on murine models of ITP using various monoclonal antibod-
ies (such as TER-119) to determine their efficacy and underlying mechanism
(Lazarus 2013).
5.3.1 Rozrolimupab
In 2013, Symphogen (a Danish biotechnology company) manufactured a new thera-

peutic product as a potential replacement for anti-D. This therapeutic product,
known as rozrolimupab, consists of 25 different monoclonal antibodies directed
against the D antigen. A monoclonal blend, rozrolimupab, is made up entirely of
human IgG1 antibodies that recognize multiple epitopes on the RhD antigen. A
Phase I/II dose-escalation study conducted across multiple centers with 61 adult,
nonsplenectomized RhD+ patients with chronic ITP has demonstrated that rozro-
limupab has an efficacy and safety profile similar to that of other plasma-derived
immunoglobulins (Robak et al. 2012). These findings provide evidence that if not a
single monoclonal antibody, at the very least a multiple monoclonal antibody blend
is able to successfully ameliorate ITP. These results also indicated that the other IgG
isotypes present in polyclonal anti-D may not be essential for ITP amelioration.
Polyclonal anti-D consists primarily of IgG1 antibodies, as used in rozrolimupab,
but it also contains a smaller fraction of IgG2, IgG3, and IgG4 antibodies. Although
the results of the study indicated that IgG1 itself is sufficient for ITP amelioration,
the potential effects of the addition of other isotypes of IgG to rozrolimupab are not
known (Lazarus 2013).
There are several benefits for using rozrolimupab as opposed to polyclonal anti-
D. Most importantly, unlike donor-derived anti-D, rozrolimupab can be manufac-
tured in vitro without any anticipated shortage of quantity and with a significantly
lower risk of potentially transferring pathogens between individuals (Lazarus 2013).
The efficacy of rozrolimupab has only been demonstrated in one study so far, and
responses observed in further studies will help decide its future implications in clin-
ical use.
ITP Patient ITP Patient injected with anti-D
Fig. 5.1 Mononuclear phagocytic system blockade as a potential mechanism of action of anti-D
in ITP. Left: In ITP, platelets are opsonized by anti-platelet antibody. Opsonized platelets then bind
to FcγRs on macrophages leading to platelet phagocytosis thus decreasing platelet counts. Right:
Anti-D binds to RhD+ RBCs and these opsonized RBCs outcompete opsonized platelets, thus
sensitized RBCs occupy a greater number of FcγRs present on the surface of the macrophages (i.e.
competitively inhibiting sensitized platelets from binding to the macrophage FcγRs). The macro-
phages phagocytose the bound RBCs leading to a decreased RBC count in some patients while
platelet levels are elevated. Macrophages; FcγR; Platelet; Platelet surface antigen;
RBC; RhD antigen; Anti-platelet antibody; Anti-D
Conclusion
Anti-D is a polyclonal immunoglobulin (IgG) directed against the D antigen pres-
ent on human red blood cells and is used clinically to treat ITP and prevent
HDFN. As a donor-derived product, it is present in limited quantities and carries
the theoretical risk of potentially transferring pathogens. Coupled with the fact
that the first priority for anti-D usage is in preventing HDFN, there is incentive
present for developing a replacement to be utilized for ITP treatment. However
the mechanism of action of anti-D in ITP remains unclear. To date, research has
proposed MPS blockage, cytokine modulation, and the presence of idiotypic anti-
bodies to contribute to the potential mechanisms. The discovery and efficacy of
rozrolimupab, a monoclonal blend of anti-D, have provided support against idio-
type antibodies as a potential mechanism of action and proved that it may be pos-
sible to replace polyclonal anti-D with a blend of multiple monoclonal
recombinants. Building on the information already present, further research can
help elucidate the mechanism of anti-RBC antibodies which can then be utilized
to synthesize a replacement recombinant antibody for clinical use.
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Mechanisms of Action
and Immunomodulation by IVIg 6
Alan H. Lazarus
6.1 Introduction
Autoantibodies represent a significant presence in mediating tissue damage in a

multitude of autoimmune syndromes. In many of these diseases, the autoantibodies
exert their inflammatory effect through activating Fc receptors for IgG (FcγRs), a
well-established class of immune cell surface receptors that interact with the Fc
domain of IgG. There are four classes of activating FcγRs: FcγRI which binds non-
complexed IgG with high affinity and FcγRIIA, FcγRIII, and FcγRIV which are
known as low-affinity receptors and bind complexed IgG.
FcγRIIA is found in humans but not mice, while FcγRIV exists in mice but not
humans. There is also an Fc receptor known as an inhibitory receptor (FcγRIIB)
which exists in both mice and humans. Although this last receptor is known as an
inhibitory receptor, it is nevertheless capable of mediating clearance of soluble
immune complexes in the liver (Ganesan et al. 2012). Conversely although FcγRIII
is well known as an activating receptor, it also can mediate inhibitory signalling
controlling inflammatory responses (Aloulou et al. 2012). IVIg anti-inflammatory
activity has been implicated in disease models where autoantibodies and Fc recep-
tors play a pivotal role in the autoimmune pathophysiology.
IVIg was originally used as a replacement product for those deficient in
IgG. However, with the appreciation that IVIg has immunomodulatory effects in
autoimmunity, its use has greatly expanded. Some of the challenges with clearly
A.H. Lazarus, PhD

Centre for Innovation at The Canadian Blood Services, Ottawa, ON, Canada
Toronto, ON, Canada
Departments of Medicine and Laboratory Medicine & Pathobiology, Faculty of Medicine,

74 A.H. Lazarus
understanding the mechanism of action of IVIg in autoimmunity are that we have

an incomplete knowledge of autoimmune pathophysiology particularly in patients.
Studies in animal models have predominated as tools in attempting to unravel the
mechanisms of IVIg action. Even among well-studied murine models such as the
krn model of rheumatoid arthritis (Kouskoff et al. 1996), the immune pathophysiol-
ogy is often more complex than we originally anticipated. To appreciate the role of
IVIg in ameliorating autoimmune disease activity, it becomes critically important to
understand the pathophysiology which we are attempting to reverse with IVIg. For
this reason, a passive model of immune thrombocytopenia (ITP) has been favoured
for understanding IVIg action. Part of the reason for selecting murine ITP as a
model to understand IVIg action is that it is clear that the majority of patients with
ITP in fact benefit from IVIg therapy. This contrasts with other animal models often
used to understand IVIg action including murine models of rheumatoid arthritis or
multiple sclerosis where the corresponding human diseases do not fare as well or as
consistently as IVIg responses in ITP.
One of the strengths of the passive murine ITP model include that we can control
the antibody causing thrombocytopenia. Available are a variety of monoclonal anti-
platelet antibodies which mediate thrombocytopenia in mice. Some of these anti-
bodies mediate the thrombocytopenia in a manner dependent on activating Fc
receptors (Li and Kimberly 2014). In fact some monoclonal antibodies can specifi-
cally cause thrombocytopenia through a selected activating Fc receptor such as via
FcγRIII or via FcγRIV (Yu et al. 2016). In addition, monoclonal antiplatelet anti-
bodies are available that cause thrombocytopenia independent of any activating Fc
receptor (e.g. anti-GPIb), and in this case IVIg anti-inflammatory activity does not
affect the thrombocytopenia (Webster et al. 2006; Li et al. 2015). There is further an
available immune active model of murine ITP where we can control the thrombocy-
topenia mediated by either humoral immunity or T cell-mediated immunity. In the
active ITP model, IVIg anti-inflammatory activity was evident when the thrombo-
cytopenia was caused by humoral immunity but not by T cell-dependent active ITP
(Chow et al. 2010). Thus the murine thrombocytopenia models have contributed
greatly to our understanding of IVIg mechanisms.
6.2 Anti-idiotype Antibodies
Anti-idiotype antibodies are found in normal healthy individuals and are antibodies
that can bind the antigen-combining region of pathogenic antibodies and neutralize
them. One of the concepts in autoimmunity is that the normal repertoire of a patient’s
antibodies is dysregulated which contributes to autoimmune tissue destruction.
Since a normal repertoire of anti-idiotype antibodies would be expected in IVIg, it
was hypothesized that this could explain IVIg anti-inflammatory effects (Table 6.1).
IVIg has been known to display anti-idiotype antibodies relevant to a number of
diseases including lupus, pemphigus vulgaris, antiphospholipid syndrome and other
diseases. Early work demonstrated that IVIg contains antibodies capable of neutral-
izing the activity of autoantibodies directed against the major platelet autoantigen,
6 Mechanisms of Action and Immunomodulation by IVIg 75
Table 6.1 Selected potential Anti-idiotype antibodies

mechanisms of IVIg Mononuclear phagocytic system blockade
immunomodulation in ITP
Cytokine modulation
and other diseases
Neonatal Fc receptor
IgG Fc region sialylation
Dendritic cell activity
T regulatory cell activity
Immune complex formation
Antibody dimers/multimers
Apoptosis
Complement
Effects on B cells
Other mechanisms
glycoprotein (GP) IIb/IIIa (Berchtold et al. 1989). Conversely, however, other work
found IVIg to be ineffective in autoantibody neutralization in patients with ITP
(Barbano et al. 1989). In murine and rat models of passive ITP, studies from two
different groups showed that anti-idiotype antibodies were not necessary for disease
amelioration (Crow et al. 2001; Hansen and Balthasar 2002).
Thus at least in ITP, the contribution of anti-idiotype antibodies to IVIg activity
could be questionable.
It is important to note that IVIg has efficacy in a large number of different auto-
immune diseases and inflammatory states. While it would be nice, or convenient, to
consider that IVIg may have the same mechanism of action in all diseases, it is
equally possible that IVIg works by different mechanisms in different diseases.
Recent data in chronic inflammatory demyelinating polyneuropathy (CIDP) patients
has shown that following IVIg administration, there is an increase in the measurable
number of IgG dimer levels in some responding patients. Although it is certainly
possible that the formation of these IgG dimers could mediate IVIg activity by
mechanism not involving autoantibody neutralization (discussed in the immune
complex section of this review), the IgG dimers formed in the patients after admin-
istration of IVIg actually contained antibodies with reactivity against peripheral
nerve fibres (Ritter et al. 2015). These results allowed the authors to speculate that
the immune complexes formed following IVIg administration in these patients
likely included autoantibodies from the patients and anti-idiotype antibodies from
the IVIg. Whether neutralization of the autoantibodies is the mechanism underlying
IVIg anti-inflammatory effects in CIDP remains to be explored but is certainly a
mechanism worthy of exploring further in this disease and perhaps others.
6.3 Mononuclear Phagocytic System (MPS) Blockade
In ITP, the site of platelet clearance is largely considered to be the spleen and liver
which are organs that also host the MPS (previously referred to as the reticuloendo-
thelial system). In patients with autoantibodies directed to the major platelet
76 A.H. Lazarus
autoantigen (GPIIbIIIa), phagocytic platelet destruction by the MPS has long been
considered to be the predominant mechanism contributing to the thrombocytopenia.
Early direct experiments that MPS blockade by IVIg could rescue antibody-
opsonized cells from phagocytosis were experiments by Fehr et al. (1982) who
demonstrated that in patients with ITP who were not splenectomised, that IVIg
treatment prolonged the in vivo clearance of antibody-opsonized erythrocytes.
These results have been confirmed by others (reviewed in; Crow and Lazarus 2008).
The first report of the treatment of an autoimmune disease with IVIg was by
Imbach et al. (1981) who demonstrated that IVIg improves ITP. Salama then noticed
that IVIg caused anaemia and postulated that the success of IVIg in treating ITP was
due to IVIg binding erythrocytes and competitively inhibiting macrophages in the
MPS by the sensitized erythrocytes (Salama et al. 1983). One year later Salama and
colleagues showed that anti-D could treat ITP in RhD-positive patients (Salama
et al. 1984). This elegant hypothesis and clinical success firmly cemented in the
concept that IVIg and anti-D both work by competitively blocking the MPS, and
most ITP-treating physicians have used IVIg and anti-D interchangeably in RhD+
patients.
Clarkson and colleagues also showed that direct inactivation of the MPS using
antibodies that bind and block activating Fc receptors were also able to ameliorate
ITP (Clarkson et al. 1986). In the passive murine ITP model, recent work has shown
that a monomeric antibody-fusion protein which binds and blocks activating Fc
receptors was also able to ameliorate the thrombocytopenia (Yu et al. 2016).
Although MPS blockade is an accepted theory to explain the mechanisms of
action of IVIg in ITP, some patients have responded to F(ab′)2 fragments of IVIg
which essentially have no Fc receptor binding activity (Tovo et al. 1984; Burdach
et al. 1986). The original concept behind MPS blockade was that MPS blockade is
due to a direct competitive mechanism whereby opsonized platelets are competing
with IVIg which bound to an antigen in a patient. However, this is not the only
potential mechanism of MPS blockade; another possibility includes that IVIg-
opsonized erythrocytes, once phagocytosed, poison the macrophage by the release
of iron. Although it seems likely that inhibition of the MPS likely underlies at least
some of the activity of IVIg, further work is needed.
6.4 Cytokine Modulation
A number of studies have examined patient’s pre-and post-IVIg administration to

look for changes in cytokine levels commensurate with disease amelioration.
Changes in interleukin (IL)- 6, IL- 8, tumour necrosis factor (TNF) α and IL-1
receptor antagonist were found increased after 1 h of IVIg administration (Aukrust
et al. 1994). Increases in IL-10 2 h post-IVIg administration and monocyte chemo-
tactic protein-1 (MCP-1) 7 days posttreatment were observed by Cooper et al.
(2004). Work from the laboratory of Donald Branch and colleagues suggested that
IL-11 may explain IVIg activity in experimental autoimmune encephalomyelitis
(Figueiredo et al. 2014), while work from Ravetch and colleagues suggested that
IL-33 (Anthony et al. 2011) or CSF-1 (Bruhns et al. 2003) may explain IVIg activity
in murine models of autoimmunity. Work from the author’s laboratory using mice
separately deficient for the IL-1 receptor, IL-4, IL-10, IL-12β, TNFα, interferon γ
receptor and macrophage inflammatory protein (MIP)1α showed that IVIg could
still ameliorate murine ITP demonstrating that the presence of these individual cyto-
kines is not critical in the amelioration of thrombocytopenia (Crow et al. 2007). In
addition IVIg worked normally in mice deficient for the common cytokine receptor
γ chain (an immune signalling component required for signal transduction through
the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) in the same study. In addi-
tion, work from Aubin and colleagues showed that mice exposed to IVIg displayed
no modulation of messenger RNA for 84 different cytokines, chemokines, or recep-
tor messenger RNAs (Aubin et al. 2007). Recent data has shown that IVIg can
upregulate the expression of the MiR-146a microRNA controlling cytokine signal-
ling pathways in human monocytes affecting the expression of several cytokines
(Loubaki et al. 2017).
A recent study performed in 11 patients with CIDP treated with IVIg interest-
ingly showed decreases in mRNA from blood cells for three genes related to the
TNFα receptor 1 (TNFR1) pathway in patients who responded to IVIg (Richard
et al. 2016). In this study, the authors analysed pretreatment levels of each mRNA
with responses 3 weeks after the initiation of IVIg therapy.
Some of the challenges with assessing the contribution of different cytokines to
IVIg action include the time of cytokine analysis, whether cytokines are being
examined from plasma or a cell fraction, and in mRNA studies whether these tran-
scripts actually result in the release of the cytokine. In addition, there is always the
possibility that changes in a mRNA from a particular cellular subset may be over-
shadowed by other subsets present in the sample. Again it cannot be overempha-
sized the additional possibility that IVIg works by different mechanisms in different
diseases.
6.5 Neonatal Fc Receptor (FcRn)
IgG has a long half-life in serum, and this is largely contributed to by the activity of
FcRn which binds IgG and protects it from intracellular degradation allowing it to
be trafficked out of the cell (Roopenian and Akilesh 2007). IVIg has been shown to
accelerate the clearance of pathogenic antibodies (Li et al. 2005). Because IVIg is
given at a very high dosage, it has the capability of saturating the FcRn and acceler-
ating the clearance of IgG molecules from the host. The accelerated clearance of
IgG from the host would include all antibodies including those that are pathogenic
as well as the IVIg itself.
To directly address the hypothesis of the contribution of FcRn activity to IVIg
effects in ITP, we used two different models of FcRn genetically-deficient mice and
clearly showed that IVIg ameliorated antiplatelet antibody-mediated thrombocyto-
penia in the absence of FcRn (Crow et al. 2011). These results demonstrated that
IVIg is not dependent on FcRn expression for its ameliorative effect in acute murine
78 A.H. Lazarus
passive ITP. Although it is possible that in other diseases there is a role for FcRn
activity in IVIg action, this could be unlikely given our results in the ITP model. An
additional complexity for FcRn in mediating the effects of IVIg in ITP is that IVIg
effects can be seen in patients within 1 or 2 days, whereas the ability of FcRn to
significantly deplete autoantibodies generally tends to take longer. For this reason
we do not favour the FcRn hypothesis for explaining IVIg effects.
6.6 I gG Fc Region Sialylation and the Inhibitory Fc Receptor

(FcγRIIB)
Perhaps one of the most controversial areas of IVIg mechanisms is in the area of
IgG Fc region sialylation. IgG molecules have an asparagine amino acid corre-
sponding to position 297 on the Fc region of IgG which is occupied by N-linked
glycosylation. The glycan structure at this position generally determines the ability
of the IgG to interact with Fc receptors as well as activate complement. Different
glycan structures at this position can increase or decrease the ability of the IgG to
interact with Fc receptors generating antibodies that can have altered inflammatory
activities (Schwab and Nimmerjahn 2013). In the case of IVIg, it was hypothesized
that an altered glycan structure could also mediate anti-inflammatory activity. This
anti-inflammatory activity was shown to be mediated by the presence of a terminal
sialic acid on the glycan structure. A number of high-impact papers published on
this hypothesis have demonstrated a very specific anti-inflammatory pathway
through which these sialic acid-containing IgG molecules mediate immunomodula-
tory effects in autoimmunity. The pathway was demonstrated to commence by the
sialic acid expressing IgG Fc region interacting with a molecule on the surface of
dendritic cells called dendritic cell-specific intercellular adhesion molecule-3-
grabbing non-integrin (DC-SIGN) in humans. Stimulation of this pathway then led
to the involvement of a number of intermediates including IL-33, basophils and the
inhibitory Fc receptor (FcγRIIB) mediating anti-inflammatory activity. Although
several investigators have shown support for this theory, a much larger number of
investigators have addressed various aspects of this pathway and not found support
for the hypothesis. For those who are interested, readers are referred to reviews
which discuss this hypothesis from a positive perspective (Anthony and Ravetch
2010; Schwab and Nimmerjahn 2013) as well some which discuss the concept less
favourably (von Gunten et al. 2014; Crow and Lazarus 2016).
6.7 Dendritic Cells (DC) and Immunomodulation
DC play a central role in the priming of adaptive immune responses, yet they can
also tolerize peripheral CD4+ and CD8+ T cells, and DC have been viewed as
promising targets for immunotherapy. IVIg has been known to inhibit T-cell prolif-
eration and T-cell cytokine production, but the mechanism of how IVIg could medi-
ate these effects was originally unknown. IVIg was then shown to inhibit the
maturation of DC and modulate their activation and survival, possibly resulting in

abrogation of T-cell activation and proliferation (reviewed in; Crow et al. 2009). DC
involvement in IVIg activity at that time was poorly understood. Were the DC
inflammatory or anti-inflammatory? How did IVIg affect these DC? Were DC
involved directly or indirectly with IVIg? What was the active component within the
IVIg preparation that affected these DC?
These questions were addressed when it was shown that IVIg action was likely
the result of the formation of IgG immune complexes in the recipient (Siragam et al.
2005) and that these immune complexes likely bound to activating Fc receptors on
the surface of DC triggering their anti-inflammatory activity (Siragam et al. 2006).
An additional possibility was that there were IgG dimers in the IVIg product
(Teeling et al. 2001) or that IVIg formed dimers or multimers soon after in vivo
injection and that these perhaps bound activating Fc receptors on the DC. Although
all of the downstream elements of this pathway have yet to be discovered, this path-
way has been well supported by the work of others in several disease models
(reviewed in; Crow et al. 2009).
DC are potent antigen presenting cells that are well-positioned to stimulate the
production of T regulatory cells (Trinath et al. 2013) as well as initiate other anti-
inflammatory pathways. In fact, a number of studies have shown that IVIg stimu-
lates the production of T regulatory cells that have activity not just in ITP but also
other disease models (Massoud et al. 2017). Although the complete linkage between
DC and T regulatory cells has not yet been confirmed, IVIg induces the induction of
a cyclooxygenase-2-dependent prostaglandin E2 pathway in DC (Trinath et al.
2013), and recent studies have shown that IVIg specifically induces an upregulation
in tolerizing dendritic cells in the spleen (Kapur et al. 2016). Some of the other
recent immunomodulatory effects downstream of IVIg administration include
increases in the appearance of myeloid-derived suppressor cells which can also shed
light on some of IVIgs immunomodulatory effects (Aslam et al. 2017).
6.8 I mmune Complexes, IgG Multimers, and Potential

Future Therapeutics
IVIg can potentially bind to a number of different particulate or soluble antigens,

and different antibodies within IVIg may be responsible for different therapeutic
effects through a variety of mechanisms. As indicated above, early work showed
that IVIg could contain dimers or multimers which appeared to cause side effects in
some patients. Later work then showed that the IgG dimers present in IVIg could
potentially mediate IVIg activity in murine passive ITP (Teeling et al. 2001).
We speculated that perhaps IVIg, upon injection into a patient, could also form
small molecular weight immune complexes which could bind and trigger anti-
inflammatory activity through activating Fc receptors on dendritic cells. The first
step in this process showed that IgG targeted to a soluble antigen could recapitulate
the effects of IVIg in murine ITP (Siragam et al. 2005). We observed that antibodies
forming immune complexes with an experimental antigen (ovalbumin) as well as
80 A.H. Lazarus
endogenous mouse albumin or mouse transferrin could all mediate IVIg-like activ-
ity in both murine ITP and in a murine model of arthritis. Because these immune
complexes worked at a 1000-fold lower dose than IVIg, this gave rise to the concept
that IVIg could potentially be replaced with either an immune complex or a modi-
fied “synthetic immune complex”. The advent of molecular engineering and the
ability to create IgG Fc fusion proteins consisting of multiple Fc regions linked to
each other has given rise to a number of different potential IVIg recombinant
replacements.
Multimerized IgG Fc products fall into a number of different categories and have
been recently discussed and compared in detail (Zuercher et al. 2016). One type of
IgG multimer, called a Stradomer has used fusion of the human IgG2 hinge region
to a human IgG1 Fc, allowing the IgG1 Fc fragment to form multimers. These
Stradomers have protective effects in murine passive ITP, collagen-induced arthri-
tis, a mouse model of myasthenia gravis and an experimental autoimmune neuritis
model (Niknami et al. 2013). Hexa-Fc or HexaGard was selected to oligomerize
monomeric Fc into well-defined hexameric oligomers capable of binding to high-
affinity Fc receptors (Czajkowsky et al. 2015). Another molecule called SIF3 is a
trimeric Fc molecule where the Fab portions of IgG have been substituted with
human IgG Fc fragments and has anti-inflammatory activity in the passive murine
ITP model and murine arthritis. In addition, a number of other similar strategies are
available and have recently been discussed (Zuercher et al. 2016).
6.9 Other Mechanisms
In addition to the mechanisms and therapeutics discussed in this review, there are a
number of other potential mechanisms of IVIg action including the ability of IVIg
to affect programmed cell death or apoptosis and the ability of IVIg to mediate
effects in conjunction with complement and effects on B cells. Although IVIg is
used worldwide to treat a multitude of different autoimmune syndromes, its
mechanism(s) of action remain unresolved. The push to replace IVIg with a mono-
clonal antibody will hopefully alleviate many of the problems with IVIg and give
rise to a more effective product for patients.
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Immunomodulatory Drugs
and Monoclonal Antibodies 7
Howard A. Liebman
7.1 Immunity: Recognition of Nonself
The traditional classification of the human immune system divides the host response
to external pathogens and transformed cells into innate immunity and adaptive
immunity (Parkin and Cohen 2001). The innate immune system is comprised of
neutrophils, monocytes, macrophages, dendritic cells, NK lymphocytes, and the
plasma complement proteins. More recent definitions have expanded the innate
immune system to include platelets, endothelium, and the coagulation cascade
(Parkin and Cohen 2001; Blumberg et al. 2009; Mantovani et al. 1997; Loof et al.
2011). The innate immune system represents the first line of host defense against
foreign pathogens. Innate immunity performs its immune surveillance function via
Toll-like receptors which recognize conserved pathogen-associated molecular pat-
terns (PAMPS) but also pattern-recognition receptors (PRRs) and NK receptors
(Kawai and Akira 2011). The initial response of the innate immune systems feeds
into and directs the subsequent responses of adaptive immunity by antigen process-
ing and cytokine production (Parkin and Cohen 2001).
Adaptive immunity involves the expression of a targeted lymphoid response
against foreign pathogens involving thymic (T) lymphocytes and antibody-
producing B lymphocytes. The targeted responses of the adaptive immune system
can further recruit and amplify the cellular responses of the innate immune system
(Parkin and Cohen 2001). This is performed in large part by antibody-mediated
pathogen clearance. Antibodies can also mediate complement lysis of pathogens in
H.A. Liebman, MA, MD

Jane Anne Nohl Division of Hematology and Center for the Study of Blood Diseases, Keck
School of Medicine of the University of Southern California, Los Angeles, CA, USA
Norris Cancer Center, 1441 Eastlake Ave, Rm 3466, Los Angeles, CA 90033, USA
e-mail: [email protected]; [email protected]

86 H.A. Liebman
addition to Fc-receptor phagocytosis of pathogens by neutrophils, monocytes, mac-

rophages, and hepatic Kupffer cells. Also cytokines and immune-modulatory mol-
ecules produced by lymphocytes can activate and upregulate innate immune
responses by activation of subtypes of monocytes and macrophages (Gordon and
Taylor 2005). Cellular immune responses of the cytotoxic (CD8+) T lymphocytes
can clear viral infected and transformed cells. Both antibody and cellular immune
responses are regulated by antigen-specific helper/inducer (CD4+) T lymphocytes.
The CD4 lymphocyte can also function as an effector cell, enhancing the intracel-
lular macrophage killing of pathogens by the production of interferon-γ (Gordon
and Taylor 2005).
7.2 Autoimmunity: Loss of Self-Tolerance
During the development of mature B and T cells, lymphoid precursors highly

attracted to self-antigens are eliminated. Lymphocytes in the bone marrow will
become B cells and those that enter the thymus will become T cells. As they are
maturing, those cells that are self-reactive will undergo apoptosis, in a process that
is called central tolerance. Most of the deletion of strongly self-reactive T lympho-
cytes occurs in the thymus (Palmer 2003; Sakaguchi 2004).
Most of the lymphocytes that recognize nonself (foreign) antigens will, there-
fore, enter the peripheral circulation occupying lymph nodes, spleen, and bone mar-
row where they will expand when they meet antigen. A few self-recognizing
lymphocytes with low reactivity to self will also enter the peripheral circulation,
where they will either remain inactive or be deleted when activated in order to pre-
vent disease. This is called peripheral tolerance (Sakaguchi 2004; Takahashi and
Nomura 2003). Peripheral tolerance is mediated in large part by natural and induc-
ible T regulatory cells (Treg) (Gordon and Taylor 2005; Palmer 2003; Sakaguchi
2004; Takahashi and Nomura 2003).
The existence of families with multiple individuals with autoimmune disorders,
an increased risk of autoimmune disorders in siblings, and the pronounced increased
risk of such disorders in identical twins strongly speaks to a genetic propensity for
autoimmune diseases (Kuchroo et al. 2012; Gregersen and Behrens 2006). Murine
and human genome-wide studies have found a number of genes associated with an
increased risk of autoimmunity. While some genes are well known to be associated
with immune regulation, genes in the major histocompatibility complex (MHC)
region account for the greatest number of genetic associations with autoimmune
disease (Rioux et al. 2009). Such changes in the MHC genes may account for the
failure to delete some lower affinity self-reacting lymphocytes which can escape
central deletion.
Environmental factors undoubtedly contribute to the development of autoim-
munity. Even in identical twins, the incidence of autoimmune disorders is only
40–50% of the monozygotic sibling (Selmi et al. 2004). There are a number of
environmental factors that have been shown capable of inducing autoimmune
responses in genetically susceptible individuals. Epidemiologic studies have
7 Immunomodulatory Drugs and Monoclonal Antibodies 87
found associations between acute and chronic viral or bacterial infections,

immunizations, environmental toxins, certain drugs, smoking, vitamin D defi-
ciency, and even changes in the intestinal microbiome to potentially induce
autoimmune disorders (Kuchroo et al. 2012; Gregersen and Behrens 2006;
Kosiewicz et al. 2014).
The innate immune system also plays an important role in maintaining self-
tolerance. However, an aberrant innate immune response to pathogens, drugs, tox-
ins, or commensal microbiota can induce a severe inflammatory state presenting the
adaptive immune system with autoantigens derived from damaged tissues and
inducing an adaptive T- and B-lymphocyte autoimmune reaction. Under such cir-
cumstances the presence of inflammatory-induced co-stimulatory cytokines such as
interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) further drives the adaptive
immune response by inhibiting Treg suppressor function leading to a chronic autoim-
mune state (Kuchroo et al. 2012; Gregersen and Behrens 2006; Dissanayake et al.
2011).
In susceptible individuals, the induction of autoimmunity pushes the
T-lymphocyte repertoire toward a pro-inflammatory state defined by a pattern of
cytokines expressed by the subsets of thymic lymphocytes (Kuchroo et al. 2012;
Gregersen and Behrens 2006). The inflammatory TH1 CD4+ helper lymphocyte rep-
ertoire is defined by the production of interferon-γ, interleukin 2 (IL-2), and
TNF. The TH17 lymphocyte repertoire is defined by the secretion of IL-17 and
IL-22. A third pro-inflammatory repertoire termed TFH lymphocytes expresses the
CD4+ inducible T-cell co-stimulator receptor and the C-X-C chemokine 5 receptor
and secretes IL-21. TFH lymphocytes support the expansion of autoantibody produc-
tion. A TH2 repertoire expresses the anti-inflammatory cytokine Il-10, along with
IL-4, IL-5, IL-6, IL-9, and IL-13, which can invoke strong antibody responses and
inhibit some neutrophil and macrophage effector functions (Del Prete 1998).
Suppression of the pro-inflammatory lymphocyte repertoires requires reconstitution
of Treg suppressor function (Kuchroo et al. 2012; Gregersen and Behrens 2006;
Sakaguchi et al. 2006).
The challenge for the clinician is to modulate the immune system in patients
with autoimmune disease resulting in a therapeutic induced self-tolerance with-
out inducing significant immune suppression, therefore, diminishing the patient’s
immune response to foreign pathogens and transformed cells. The heterogeneity
of the underlying pathophysiology may account for the variable responses to
many treatment regimens. In addition, the process of immunomodulation can in
and of itself result in further disruption of immune tolerance resulting in the
emergence on new autoimmune disorders. This can occur with immune suppres-
sion in patients with bone marrow or solid organ transplants (Minchinton and
Waters 1985; Taylor et al. 2006). The recent use of the cytotoxic anti-CD52
monoclonal antibody, alemtuzumab, in multiple sclerosis has resulted in a sur-
prisingly high incidence of autoimmune disorders suggesting that imbalance in
T-lymphocyte recovery after significant lymphocyte depletion can lead to a loss
of self-tolerance (Cuker et al. 2011; Daniels et al. 2014; Von Kutzleben et al.
2016; Jones et al. 2013).
88 H.A. Liebman
7.3 Therapeutic Approaches to Immune Modulation
The development of immune-modulating therapies for induction of self-tolerance

with transplantation and treatment of autoimmune disorders has progressed rapidly
since the first reports by Schwartz and Dameshek on the induction of immune toler-
ance in rabbits with 6-mercaptopurine (Schwartz and Dameshek 1959, 1960). Only
4 years after their initial reports, they subsequently reported on the use of the drug
for treatment of patients with autoimmune hemolytic anemia (Schwartz and
Dameshek 1962). Most often designated as immunosuppress drugs, subsequent
therapeutic agents have been developed that target one or more components of the
immune system. They can be broadly defined as inhibitors of T-lymphocyte activa-
tion and proliferation, inhibitors of B-lymphocyte activation and proliferation,
inhibitors of the innate immunity, inhibitors of immune cell trafficking, cytokine
inhibitors, and drugs that deplete T or B lymphocytes or both (Table 7.1). In addi-
tion they can also be classified as cytotoxic drugs used to deplete broad or specific
Table 7.1 Targets of immune-modulating agents

Cytokine and
Monocyte, Inhibitors of cell cytokine
T lymphocyte B lymphocyte macrophage trafficking receptor inhibitors
Corticosteroids Corticosteroids Corticosteroids Corticosteroids Corticosteroids
6-Mercaptopurine IVIG IVIG IVIG IVIG
Azathioprine 6-Mercaptopurine 6-Mercaptopurine Natalizumab TNF inhibitors
Mycophenolate Azathioprine Azathioprine Crizanlizumaba Infliximab
Cyclophosphamide Mycophenolate Mycophenolate GMI1070b Adalimumab
Alemtuzumab Cyclophosphamide Cyclophosphamide Golimumab
Cyclosporine Alemtuzumab Fostamatinibc Certolizumab
Tacrolimus Rituxumab IL-1 inhibitors
Sirolimus Ofatumumab Anakinra
Everolimus Veltuzumab Canakinumab
BI655064d Ocrelizumab Rilonacept
BMS98004d IL-12/23
inhibitors
Ustekinumab
IL-6 inhibitors
Tocilizumab
IL-17 inhibitors
Secukinumab
Ixekizumab
Brodalumab
BAFF inhibitors
Belimumab
a
Crizanlizumab is a P selectin inhibitor in trial
b
GMI 1070 is an E-selectin inhibitor in trial
c
Fostamatinib is a Syk inhibitor in clinical trial
d
BI655064 and BMS986004 are CD40 ligand inhibitors in clinical trial
cellular populations or agents that inhibit signaling pathways which can mediate
their effect by blocking extracellular or intracellular targets. The contemporary rep-
ertoire of immune-modulating agents has expanded from such cytotoxic chemo-
therapeutic agents as cyclophosphamide, methotrexate, and 6-mercaptopurine to
now include humanized monoclonal antibodies that either are lymphocyte cyto-
toxic, cytokine inhibitory, and cytokine receptor inhibitory or block important traf-
ficking molecules for neutrophils, monocytes, or lymphocytes. In addition, there are
an expanding number of small molecules that inhibit important intracellular signal-
ing pathways in lymphocytes. These can be cytotoxic for selected lymphocyte cel-
lular populations or downregulate lymphocyte cellular responses to activating
signals.
7.4 Glucocorticosteroids
Glucocorticosteroids (GC) were the first (Hench et al. 1949) and remain one of the
most important agents for the treatment of autoimmune disorders with the broadest
spectrum of immune-modulatory effects (Van der Goes et al. 2014). The spectrum
of their biologic effects can change with increasing doses. At low doses they affect
immune cell trafficking, downregulate FcR expression on macrophages and neutro-
phils, and modulate cell signaling. With higher doses they can induce apoptosis of
eosinophils and mast cells, inhibit dendritic cell differentiation, and suppress cyto-
kine production from T lymphocytes, macrophages, endothelial cells, and smooth
muscle cells. At very high doses, they can induce apoptosis of plasma cells and T
and B lymphocytes. Corticosteroids can inhibit B-lymphocyte differentiation into
plasma cells (Yan et al. 2015). Long-lived T-lymphocyte memory cells appear to be
resistant to the cytotoxic effect of corticosteroids. A recently described subset of T
lymphocytes termed IL-17 producing CD4/CD8 double-negative T lymphocytes
were described in patients with Sjogren’s syndrome. When studied this subpopula-
tion of thymic lymphocytes was resistant to dexamethasone treatment when com-
pared to CD4+ and CD8+ thymic lymphocytes (Alunno et al. 2013). This and other
studies show that different subsets of T lymphocytes may have significant differ-
ences in their sensitivity to corticosteroids.
Compared to other immune-modulating agents, the onset of action by glucocor-
ticoids can be very rapid. Upon binding to the glucocorticoid receptor (GR), the
complex of the receptor with the glucocorticoid (GR/GC) is rapidly transported to
the nucleus where it binds to the glucocorticoid binding sites for transcription of
specific genes. Among such gene products are other transcription factors which
work with the GR to induce transcription of additional gene products in what is
termed a feed-forward gene regulatory loop (Meijsing et al. 2009; Psarra and Sekeris
2009; Sasse et al. 2013). In addition to its role in gene transcription, the GR/GC
complex has direct and indirect effects on mitochondrial function and as yet unex-
plained cytoplasmic cellular effects.
90 H.A. Liebman
7.5 Intravenous Immunoglobulin
Intravenous immunoglobulin (IVIG) was first shown to have immunomodulatory

effects in the treatment of immune thrombocytopenia (Imbach et al. 1981; Newland
et al. 1983). While most responses as a single agent are of short duration, longer
duration responses were reported in an early trial on the treatment of ITP with
repeated infusions for patients not candidates for splenectomy (Bussel et al. 1988).
A number of possible mechanisms for the immune-modulatory effects of IVIG have
been proposed using murine experimental models and translational studies of
treated patients (Imbach et al. 2010). It is reasonable to assume that there are several
different immune-modulatory mechanisms mediated by this complex mixture of
antibodies, inclusive of immune complexes, anti-idiotypic antibodies, cytokines,
and cellular receptors that can affect the underlying immunopathology for a variety
of autoimmune disorders (Seite et al. 2008; Blasczyk et al. 1993). Its toxicities have
been well characterized and maybe related to the patients’ age, comorbidities, the
rate of IVIG infusion, and the dose of IVIG used (Hamrock 2006). However, the
lack of uniformity in most IVIG preparations could also explain the variability in
therapeutic response. It is notable that when given in a 24-h continuous infusion
combined with platelet transfusions, it induced sustained increases in platelet counts
for refractory patients who previously failed to respond to IVIG (Chandramouli and
Rodgers 2000; Olson et al. 2016). Other chapters in this volume will cover, in
greater detail, the biologic and immune-modulatory effects of IVIG. However,
it could be generalized that IVIG, after corticosteroids, is the broadest immuno-
modulatory agent affecting both the innate and adaptive immune system (Imbach
et al. 2010).
IVIG has often been combined with other immunomodulatory agents. Not cyto-
toxic, IVIG immune-modulatory effects spare bone marrow function. It is often
combined with corticosteroids in regimens for refractory ITP and autoimmune
hemolytic anemia (Bussel et al. 1988; Boruchov et al. 2007; Barcellini et al. 2014).
When combined with corticosteroids, the combination also appears to reduce the
incidence and severity of unwanted IVIG toxicities such as infusion-associated
reactions and aseptic meningitis. In the treatment of chronic ataxic neuropathy,
IVIG has also been combined with rituximab (Loscher et al. 2013). Combination
therapy for refractory ITP patients has also incorporated vincristine and azathio-
prine (Boruchov et al. 2007).
7.6 Cytotoxic Chemotherapeutic Agents
Cytotoxic agents such as cyclophosphamide (CTX), methotrexate (MTX), azathio-

prine (AZA), 6-mercaptopurine (6-MP), and mycophenolate mofetil (MMF) all
demonstrate a broad spectrum of cellular cytotoxicity that can deplete T and B lym-
phocytes along with general bone marrow suppression. They also, in a dose-
dependent manner, inhibit bone marrow production of important effectors of the
innate immune system, neutrophils, and macrophages.
7.6.1 Cyclophosphamide
The immunologic effects of cyclophosphamide have been most extensively studied

and have documented significant dose-related immune-modulating effects. Low
doses of CTX can transiently suppress CD4+FOXP3+ T regulatory cells, shifting the
CD4 T-lymphocyte repertoire toward a TH1 inflammatory pattern (Ghiringhelli
et al. 2007). Higher doses have a broader depletion of T and B lymphocytes. With
higher doses the FOXP3+regulatory T lymphocytes and hematopoietic stem cells
are much more resistant to cyclophosphamide due to their increased expression of
aldehyde dehydrogenase (Kanakry et al. 2013). Clinically, the superiority of high-
dose intravenous CTX versus low-dose oral CTX for the treatment of autoimmune
disorders has been clearly demonstrated in clinical trials on its use for the treatment
of vasculitic disorders (de Groot et al. 2009). Complete remission was obtained with
low doses of CTX when given as intravenous pulses (de Groot et al. 2009).
7.6.2 Methotrexate
Methotrexate, a dihydrofolate reductase inhibitor, was initially developed as an anti-

neoplastic agent but is often used today as an immune modulator in rheumatoid
arthritis and other autoimmune disorders. It rapidly induces apoptosis of activated
lymphocytes. Lymphocytes in G0 or G1 phase of the cell cycle are resistant to the
drugs cytotoxic effect (Genestier et al. 1998). This selective deletion of activated T
lymphocytes may partially explain how MTX therapy in juvenile rheumatoid arthri-
tis predominantly suppresses T effector cell activity but spares T regulatory cells
(Calasan et al. 2015). Methotrexate is now most often used in combination therapy
of rheumatoid arthritis but contributes to higher response which is associated with
suppression of Th1/Th2 and Th17 phenotype with increase in Treg number and func-
tion (Lina et al. 2011).
7.6.3 Azathioprine, 6-Mercaptopurine, and Mycophenolate
Less is known about the dose-related immunologic effects of 6-MP, AZA, and MMF
on T-lymphocyte subsets. AZA, 6-MP, and MMF influence purine synthesis by
inhibiting the proliferative response of lymphocytes to activating stimuli. AZA and
6-MP inhibit the first step of de novo purine synthesis suppressing both T and B
lymphocytes. The antiproliferative effect is nonselective and can result in significant
neutropenia from bone marrow suppression at higher doses (Maltzman and Koretzky
2003). MMF is more selective for lymphocyte suppression by both inhibiting purine
synthesis and by competitive inhibition of inosine monophosphate dehydroge-
nase (IMPDH). Activated lymphocytes are highly dependent on the IMPDH sal-
vage pathway for purine synthesis (Allison and Eugui 1996). Therefore, there is less
direct bone morrow suppression and greater lymphocyte selectivity (Sollinger
1995).
92 H.A. Liebman
In randomized trials to evaluate the efficacy of AZA and MMF in prevention of

acute graft rejection after kidney transplantation, MMF has shown higher efficacy
(Merion et al. 2000). However, there is no comparative data on the use of these
agents in the treatment of autoimmune disease. Using these agents, response to
treatment may require several months of therapy. A study on the treatment of
immune thrombocytopenia (ITP) with AZA, response in some patients took up to
4 months (Quiquandon et al. 1990).
7.7 Monoclonal Antibodies
Monoclonal antibodies, targeting specific antigens on lymphocyte subsets, have

proven to be important therapeutic agents for the treatment of various autoimmune
disorders. The contemporary repertoire of therapeutic immune-modulating human-
ized monoclonal antibodies can be classified as either cytotoxic for subsets of lym-
phocytes or inhibitory of important cytokines or chemokines, their receptors, and
important cellular trafficking molecules.
7.7.1 Cytotoxic Monoclonal Antibodies
The B-lymphocyte antigen, CD20, was the first target for the development of a
humanized monoclonal antibody. This was antigen originally selected as a potential
target for treatment of B-cell lymphomas. Rituximab, the first of these humanized
monoclonal antibodies to target the CD20 antigen on B lymphocytes, has been suc-
cessfully utilized in the treatment of a number of autoimmune disorders. There are
case reports and Phase II clinical trials on its use in patients with autoimmune
hemolytic anemia, immune thrombocytopenia, coagulation factor VIII inhibitors,
thrombotic thrombocytopenic purpura, rheumatoid arthritis, vasculitis, cryoglobuli-
nemia, multiple sclerosis, and neuromyelitis optica (Dierickx et al. 2015; Patel et al.
2012; Franchini and Lippi 2008; Coca and Sanz 2012; Cacoub et al. 2012;
Rubenstein et al. 2006). However, in the United States, rituximab is only FDA
approved for the treatment of rheumatoid arthritis in combination with methotrex-
ate. It is frequently combined with corticosteroids and other immune-modulating
agents when used to treat autoimmune disorders (Bussel et al. 2014; Gupta et al.
2002). What is notable in regard to these antibody-mediated disorders is that the
pathogenic antibodies are IgG immunoglobulins, produced primarily by plasma
cells which show minimal CD20 expression. Despite this, treatment responses,
depending upon the specific immunopathic disorder, range from 20 to 80%. In ITP,
studies by Stasi and colleagues found that specific changes in the T-lymphocyte
repertoire best define those patients who obtain a complete response to rituximab
treatment compared to patients who failed to respond (Stasi et al. 2007, 2008).
Increases in CD4+FOXP3+ T regulatory cells number and function are seen in the
patients who obtain long-term complete remissions (Stasi et al. 2007, 2008). This
effect may be due to modulation or B- and T-lymphocyte cross talk or depletion of
B lymphocytes as antigen-presenting cells. The later mechanism may be favored

since anti-CD20 therapy given to adult ITP patients in the first year after diagnosis
appears to be associated with a higher rate of complete response. This was further
supported by a Phase II trial of a subcutaneous anti-CD20 humanized monoclonal
antibody, veltuzumab, which showed a higher response to patients treated in the first
year (Liebman et al. 2013). The anti-CD20 humanized monoclonal antibody, ocreli-
zumab, has recently been FDA approved for the treatment of primary progressive
multiple sclerosis, becoming only the second humanized B-cell-depleting monoclo-
nal to be FDA approved to treat nonmalignant autoimmune disorders (Montalban
et al. 2017).
Alemtuzumab is a humanized monoclonal antibody that binds to the CD52 anti-
gen present on most mature lymphocytes. It rapidly depletes both T and B lympho-
cytes and was originally approved in the United States for the treatment of refractory
chronic lymphocytic leukemia. A number of case reports and small clinical trials
have documented its use in several autoimmune disorders (Ru and Liebman 2003;
Gomez-Almaguer et al. 2010). Recently, alemtuzumab was approved in the United
States and Europe for the treatment of relapsing-remitting multiple sclerosis with
significant clinical superiority over β-interferon 1a (Cohen et al. 2012). However, an
unexpected late complication of this highly effective therapy has been the develop-
ment of a variety of autoimmune disorders. Over a third of patients develop immune
thyroid disease, most often Grave’s disease, which is distinctly different from the
pattern of thyroid disease that develops in the general population (Daniels et al.
2014; Weetman 2014). Also cases of immunopathic renal disease have been
observed which include membranous glomerulonephritis and anti-GBM antibody
disease (Goodpasture’s disease) (Clatworthy et al. 2008). In 2% of patients treated
in the clinical trials, acute decreases in platelet counts consistent with ITP were
observed, beginning 14–36 months after the last injection of alemtuzumab (Cuker
et al. 2011). These ITP cases all responded to standard ITP first-line therapies and
all appeared to develop unmaintained remissions similar to pediatric ITP patients.
The occurrence of the late development of other autoimmune disorders, despite
effective control of the patients’ multiple sclerosis, suggests that the potent lym-
phoid depletion by alemtuzumab results in a prolonged and significant defect in
peripheral immune tolerance that can persist for years after treatment.
7.7.2 N
oncytotoxic Immune-Modulating Monoclonal
Antibodies
A number of humanized monoclonal antibodies have been developed to bind to and

inhibit inflammatory cytokines or their receptors. The first initial therapeutic target
was tumor necrosis factor-alpha (TNF-α). TNF is a broad family of potent cytokines
central to systemic inflammation (Aggarwal 2003). It is produced by activated cells
of the innate immune system including macrophages, neutrophils, eosinophils, NK
cells, and mast cells. Inappropriate expression has been linked to a number of
inflammatory autoimmune disorders such as rheumatoid arthritis, Crohn’s disease,
94 H.A. Liebman
ulcerative colitis, and psoriasis (Rutgeerts et al. 2005). The first of these antibodies,
infliximab, which targets TNFα, has documented efficacy in inflammatory bowel
disease, rheumatoid arthritis, psoriasis, and ankylosing spondylitis (Aggarwal 2003;
Rutgeerts et al. 2005; Sands et al. 2004; Maini et al. 1999; Fong et al. 2016).
Golimumab, adalimumab, and certolizumab are the second, third, and fourth anti-
TNF-α inhibitory antibodies with the same general clinical indications as infliximab
(Hibi et al. 2017; Colombel et al. 2007; Weinblatt et al. 2017). Adalimumab was the
first totally humanized monoclonal therapeutic antibody, but except for this struc-
tural difference, there appear to be no significant therapeutic advantages to this anti-
body over the other two approved TNF-α inhibitory antibodies. A TNF receptor
fusion protein, etanercept, acts as a competitive inhibitor of TNF binding to its
receptor and is a therapeutic alternative to direct TNF inhibition.
The next generation of inhibitory antibodies targeted interleukin 1 (IL-1). IL-1 is
produced by cells of the innate immune system, macrophages, monocytes, fibro-
blasts, and dendritic cells. It may also be produced by endothelial cells, NK cells,
and B lymphocytes. There are 11 members of the IL-1 cytokine family, with IL-1
alpha and IL-1 beta being the most often studied (Garlanda et al. 2013). IL-1 is a
central mediator of the inflammatory response of the body against infection. It
induces expression of adhesion molecules on endothelial cells which results in neu-
trophil and monocyte adhesion to the vessel wall, rolling, and diapedesis into tis-
sues. It is also the major inducer of TNF and the febrile response to infection.
Canakinumab was the first FDA-approved humanized monoclonal directed against
IL-1 beta to treat auto-inflammatory syndromes such as cryopyrin-associated peri-
odic syndromes and more recently to treat juvenile rheumatoid arthritis (Kuemmerle-
Deschner et al. 2016; Orrock and Ilowite 2016). The antibody also has documented
efficacy in familial Mediterranean fever and other rare inflammatory syndrome
(Kucuksahin et al. 2017; Gattorno et al. 2017). Similar to the TNF receptor competi-
tive inhibitor, etanercept, interleukin 1 receptor competitive inhibitors, anakinra and
rilonacept, have also demonstrated activity in the treatment of rheumatoid arthritis
and other inflammatory disorders.
Therapeutic inhibitory humanized monoclonal antibodies inhibitory of interleu-
kin 6 (IL-6), tocilizumab; inhibitory of interleukin 17 (IL-17), secukinumab, ixeki-
zumab, and brodalumab; inhibitory of the interleukin 12/23 complex (IL12/23),
ustekinumab; and inhibitory of B-cell-activating factor (BAFF), belimumab, are
now approved for various immunopathic disorders.
Tocilizumab has demonstrated therapeutic efficacy in rheumatoid arthritis
(Teitsma et al. 2016) and giant cell arteritis (Ostrowsk et al. 2014). Secukinumab,
ixekizumab, and brodalumab have FDA approval and efficacy in the treatment of
refractory psoriasis and psoriatic arthritis (Mease 2015; Mease et al. 2016, 2017).
Ustekinumab by inhibition of IL12/23 downregulates the production of IL17 (Mease
2015). Therefore, it is not surprising that it has similar efficacy in the treatment of
psoriasis and psoriatic arthritis (Kavanaugh et al. 2016). Belimumab is the first
immune-modulating therapy approved for the treatment of systemic lupus erythe-
matosus (Lutalo and D'Cruz 2014). The antibody also shows promise in the treat-
ment of primary Sjogren’s disease (Mariette et al. 2015).
7.8 Inhibitory Drugs of T-Lymphocyte Function
Calcineurin inhibitors, cyclosporine and tacrolimus, inhibit the calcineurin-mediated

dephosphorylation the transcription factor nuclear factor of activated T cells (NF-AT),
which is necessary for interleukin (IL)-2 transcription and T-cell activation. The potent
T-lymphocyte suppression has made these drugs the primary therapeutic agents for
preventing graft rejection for solid organ and bone marrow transplants (Wiederrecht
et al. 1993). They have been used in a number of small studies for the treatment of
autoimmune disorders, but their toxicities and inability to induce long-term remis-
sions in most patients have limited their use. In refractory ITP low-dose cyclosporine
(2–3 mg/kg) in several small case series could induce remissions in 40–50% of
patients treated (Gesundheit et al. 2001; Kappers-Klunne and van't Veer 2001; Choi
et al. 2015). However, approximately 70% of patients relapse after drug withdrawal.
The addition of cyclosporine to combination regimens appears to enhance responsive-
ness in patients with refractory ITP (Choi et al. 2015).
Sirolimus and everolimus are inhibitors of the mTOR pathway through which a
number of cytokines induce cell proliferation. Sirolimus blocks the T-lymphocyte
proliferative stimulus of Il-2. However, the drug has a differential effect on
T-lymphocyte subsets and appears to have little suppressive effects on the in
CD4+FOXP3+ T regulatory cells (Shan et al. 2014). This may be an important role
of the drug in its use for the treatment of graft versus host disease following alloge-
neic bone marrow transplant. Only a few case reports and case series have been
published on the use of sirolimus for autoimmune disorders, the majority in pediat-
ric disorders (Miano et al. 2014; Chatrath et al. 2014). However, a recent report has
suggested an important role for the mTOR pathway in the pathophysiology of the
antiphospholipid antibody syndrome (Canaud et al. 2014).
7.9 Summary
The increasing number of therapeutic agents for autoimmune disorders has signifi-
cantly improved the outcomes for many patients but has resulted in only a small
number of sustained unmaintained remissions. The variability of treatment out-
comes to individual agents has clearly heterogeneous. As suggested by Cines and
colleagues, many autoimmune disorders, like immune thrombocytopenia, should
best be termed a syndrome and not a disease (Cines et al. 2009). Unraveling the
heterogeneity of the various autoimmune disorders should result in better selection
of therapeutic agents for the treatment of such patients.
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Use of Intravenous Immunoglobulin
in Neurology 8
Marinos C. Dalakas
8.1 Introduction
Intravenous immunoglobulin (IVIG), a pooled of polyclonal IgG from the serum of

thousands of donors, has been used as an anti-inflammatory and immunomodulat-
ing agent for the treatment of several autoimmune diseases. It has played a funda-
mental role in the treatment of several autoimmune neurological disorders with at
least three indications approved by regulatory agencies and with established effi-
cacy in additional neurological diseases based on controlled studies. The first indi-
cation was in 2008, for chronic inflammatory demyelinating polyneuropathy
(CIDP), followed by Guillain-Barre syndrome (in Europe and Asia) and then multi-
focal motor neuropathy (MMN) (Lunneman et al. 2015a, b; Hughes et al. 2009). In
this review, I will provide a summary on the current use of IVIG in neurological
diseases based on controlled studies, highlight the autoimmune rationale for using
IVIg based on the underlying pathophysiology, and briefly mention some of the
ongoing studies.
The clear and sometimes dramatic benefit exerted by IVIg in certain neurological
disorders has inevitably led to a rather liberal use of the drug even for diseases
where the data is weak or not evidence based, generating difficulties with insurance
carriers even for diseases with clear indications. Most importantly, we are now wit-
nessing the continuous use of IVIg for patients who may not anymore need it
because the disease has been put into remission or in a chronic stability status after
several monthly IVIg infusions. Until biomarkers of disease activity are identified,
M.C. Dalakas, MD, FAAN

Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA
Neuroimmunology Unit, Department of Pathophysiology, National and Kapodistrian
University of Athens Medical School, Athens, Greece

102 M.C. Dalakas
periodic assessments are now advocated for these patients to establish their need for
continuous immunotherapy, by tapering the IVIg dose or temporarily stopping IVIg
to ensure its judicious use for continuous chronic long-term therapy (Lunneman
et al. 2015a, b).
8.2 Evidence from Controlled Trials
8.2.1 Guillain-Barre Syndrome (GBS)
8.2.1.1 Definition and Pathophysiology

This is an acute demyelinating polyneuropathy that causes weakness or paralysis of
the limbs and respiratory muscles within 2–4 weeks from onset. Although the exact
target antigen is still unknown, molecular mimicry, antiglycolipid antibodies, T cell
sensitization, activated macrophages, and complement are the main immunopatho-
logical features of the disease (Lunneman et al. 2015a, b; Hughes et al. 2009;
Dalakas 2004; Gold et al. 2007; Yuki and Hartung 2012). In addition to the classic
demyelinating form of GBS, there are GBS variants, such as the acute axonal motor
or motorsensory forms, the Miller-Fisher syndrome or acute dysautonomia (Yuki
and Hartung 2012). Although IVIG appears to be also helpful in these patient sub-
sets, controlled studies have been conducted only in the classic sensorimotor demy-
elinating form.
8.2.1.2 Management
Based on at least two randomized trials, one dose of IVIG (5-day regimen of 0.4 g/
kg/d) was comparable to plasmapheresis (PE) in outcome measures including time
to unaided walking and discontinuation of ventilation [class I evidence]
(Sandoglobulin Guillain-Barré Syndrome Trial Group 1997). Combining IVIG with
PE or with 500 mg intravenous methylprednisolone produced no incremental
response (Lunneman et al. 2015a, b; Sandoglobulin Guillain-Barré Syndrome Trial
Group 1997). IVIg also remains the treatment of choice in childhood GBS, based on
observations attesting to a faster recovery and reduced morbidity, but controlled
studies are not available and may not be ever conducted.
GBS is a monophasic disease, but many patients remain with significant
weakness within the first month of the disease, in spite of the early initiation of
IVIg therapy. Whether a second IVIg infusion may add more benefit when
improvement has not occurred or it is inadequate 3 weeks after the first infusion
remains unclear in spite of anecdotal evidence. A controlled study assessing the
benefit of a second IVIG infusion is currently ongoing in the Netherlands. This
is immunologically justified because a small increase in serum IgG level,
2 weeks after one IVIg infusion, was independently associated with signifi-
cantly slower recovery and more disability at 6 months (Kuitwaard et al. 2009).
It is anticipated that this study will provide credence to the observation that a
low DeltaIgG 2 weeks after the initial infusion may be a sign that a higher dos-
age or a second infusion might be helpful for patients who exhibit poor
outcome.
8 Use of Intravenous Immunoglobulin in Neurology 103
8.2.2 C
hronic Inflammatory Demyelinating Polyneuropathy
(CIDP)

Chronic inflammatory demyelinating polyneuropathy (CIDP) is characterized by
slow onset (over weeks to months) weakness, areflexia, and impaired sensation.
Antibodies, activated T cells, and complement have been implicated in the cause of
the disease (Dalakas 2011). A subset of patients, accounting for 10% of total CIDP,
has IgG4 antibodies against two nodal antigens, neurofascin-155 and contactin; this
subset is important in discussing IVIg effectiveness because most of these antibody-
positive CIDP patients respond poorly to IVIg (Dalakas and Gooch 2016).
8.2.2.2 Management
Controlled studies have shown that steroids, plasmapheresis, and IVIG are equally
effective on a short-term basis (Dalakas 2011). The ICE trial, the largest ever con-
ducted in CIDP, has showed that IVIG is safe and effective not only for short-term
but also for long-term leading to the first FDA-approved indication for a brand of
IVIG (class I evidence) (Hughes et al. 2008). A strong and positive effect on quality
of life and improvement in some electrophysiological measurements were also
noted. In most patients, IVIG becomes clearly effective after 6 weeks, necessitating
the need for at least 2–3 infusions before concluding that it is ineffective. Although
IVIg is generally considered as first-line therapy based on the ICE trial, the choice
of how best to initiate therapy (choosing between prednisone, IVIg, or plasmapher-
esis which are all effective) is judged against cost, long-term side effects, patient
age, venous access, disease severity, and concurrent illnesses. Clinically, patients
more likely to respond to IVIG therapy are those with disease duration of less than
a year, a relapsing course, no difference in strength between arms and legs, and
electrophysiological signs of demyelination with conduction block (Lunneman
et al. 2015a, b). FcγRIIB expression was reported to be decreased in treatment-naïve
CIDP patients and upregulated upon clinically effective IVIG therapy suggesting
that the effect on FcγRIIB may be a factor predicting the patients more likely to
respond to IVIG (Lunneman et al. 2015a, b). Increased Fc glycosylation seems also
associated with disease remission and response to IVIg, but the evidence is not yet
strong enough to serve as a disease biomarker.
IVIg is not effective in patients who have a demyelinating neuropathy, resem-
bling CIDP, accompanied by an IgM monoclonal gammopathy with antibodies to
myelin-associated glycoprotein, based on a controlled study (Dalakas et al. 1996).
8.2.3 Multifocal Motor Neuropathy

Multifocal motor neuropathy (MMN) presents with a slow onset weakness and
muscular atrophy in the distal upper extremities, areflexia, preserved sensation, and
conduction block of the motor axons. IgM antibodies to GM1 ganglioside are seen
in up to 50% of these patients (Federico et al. 2000; Hahn et al. 2013).
104 M.C. Dalakas
8.2.3.2 Management
Unlike CIDP and GBS, MMN does not respond to steroids or plasmapheresis but
responds only to IVIG. Efficacy has been established with a number of controlled
trials, and IVIg is now FDA-approved for MMN (Hahn et al. 2013). The improve-
ment lasts from 3 to 6 weeks, requiring a new reinfusion at almost predictable time
periods. As symptoms diminish, the electrophysiologic conduction block may
resolve. Therapy starts with 2 g/kg, but the response can be maintained with a 1 g/
kg, a pattern also followed for CIDP.
8.2.4 Myasthenia Gravis

Myasthenia gravis (MG) is characterized by fluctuating weakness or fatigability of
the extraocular, bulbar, respiratory, and limb muscles. It is the prototypic autoim-
mune disease mediated by pathogenic antibodies to the acetylcholine receptors
(AChR); up to 5–7% of patients are seronegative, while another 5% have anti MuSK
antibodies (Vincent 2002).
8.2.4.2 Management
Patients with MG respond fairly well to the available therapies, such as anticholin-
esterases, steroids, or immunosuppressants. Plasmapheresis is effective for crises or
severe exacerbations. The use of IVIG in MG has been examined in randomized
trials for treating exacerbations in lieu of plasmapheresis. In two randomized trials,
IVIG was as effective as plasmapheresis at day 15 (Gajdos et al. 1997). In one of the
studies, there was no difference between patients randomized to 1 g/kg for 1 day
versus 2 g/kg for 2 days (Gajdos et al. 2005). IVIG was also superior to placebo,
14 days after therapy, in patients with moderate to severe MG and “worsening
weakness” (Lunneman et al. 2015a, b; Gajdos et al. 2012). Although IVIG may be
effective on a short-term basis, its role in the chronic management of the disease or
as a steroid-sparing drug has not yet been established, but two currently ongoing
trials are precisely aimed to address these questions. At present, IVIG may be justi-
fied in lieu of plasmapheresis for acutely worsening disease to prevent or minimize
impending bulbar or respiratory failure or prepare a weak patient for thymectomy.
IVIG may be also effective in Lambert-Eaton Myasthenic Syndrome, based on a
small placebo-controlled study, that showed a statistically significant increase in
muscle strength compared to placebo, 2–4 weeks after therapy (Bain et al. 1996).
8.2.5 Inflammatory Myopathies

The main subsets in this large family of diseases include: dermatomyositis (DM),
polymyositis (PM), necrotizing autoimmune (NAM), and inclusion body myositis
(IBM) (Dalakas 2004a; Dalakas 2015). Dermatomyositis causes proximal muscle
weakness and a violaceous rash on the face and extremities. Early deposition of
membranolytic attack complex (MAC) on the endomysial capillaries leads to capil-
lary destruction, muscle ischemia, and inflammation. Polymyositis is a rare T-cell-
mediated disease T cell Receptor clonality (O’Hanlon et al 1994), causing subacute
onset of muscle weakness; NAM is a macrophage-mediated, and possibly-antibody-
fixing, process, directed against muscle fibers that cause more severe, and often
acute, muscle weakness. IBM is a chronic disease with slowly progressive proximal
and distal weakness along with muscle atrophy caused by a combination of T-cell-
mediated cytotoxicity along with a degenerative process associated with protein
misfolding (Dalakas 2015).
8.2.5.2 Management
In a double-blind, placebo-controlled study, IVIg was effective in dermatomyositis
patients resulting in significant improvement of strength and muscle function, com-
pared to placebo, and a marked improvement of the active skin rash as shown in
Fig. 8.1 (Dalakas et al. 1993). Repeat muscle biopsies demonstrated significant
improvement in the muscle cytoarchitecture including increased muscle fiber diam-
eter, as shown in Fig. 8.2, revascularization, reduction of inflammation, interception
of complement deposition, resolution of immunopathology, and downregulation of
inflammatory mediators at the protein, mRNA, and gene level (Raju et al. 2005;
Documented effects on phagocytosis, complement, apoptosis
Example: Dermatomyositis (DM) (1)
Patients with DM before and after IVIG Therapy
Courtesy of Dalakas MC
Fig. 8.1 Two patients with refractory Dermatomyositis participating in the controlled study
(Dalakas at al 1993) after 3 months of IVIg therapy show a dramatic imrprovement in muscle
strength
106 M.C. Dalakas
Exmple: Dermatomyositis (DM) (2)
Histopathologic Changes in DM Muscles after IVIg
Before
After
Courtesy of Dalakas MC
Fig. 8.2 Muscle biopsy specimens from the same patient taken from the left arm (before) IVIg
and the right arm (after) after IVIg therapy. The number of capillaries (left)and the perifascicular
atrophy with inflammation before therapy have been completely resolved after therapy coinciding
with the resolution of muscle strength. The number of capilaries with the dilated lumen (left) were
also normalized due to neovascularization
Dalakas et al. 1993; Basta and Dalakas 1994). IVIg seems effective in some patients
with polymyositis and NAM (Dalakas 2004b; Lunneman et al. 2015a, b), but a con-
trolled study has not been performed. IVIg is ineffective in IBM based on two con-
trolled studies; it did however statistically improve the patients’ dysphagia (Dalakas
et al. 1997).
8.2.6 Stiff-Person Syndrome

Stiff-person syndrome (SPS) is a disabling autoimmune disorder characterized by
muscle rigidity, episodic muscle spasms, and antibodies to glutamic acid decarbox-
ylase (GAD65).
8.2.6.2 Management
In a placebo-controlled, crossover study, IVIG significantly decreased stiffness
scores, and substantially increased walking and functions of daily activities (Dalakas
et al. 2001). The study concluded that IVIG is effective as supplementary therapy in
patients with SPS who do not adequately respond to first line drugs, such as diaze-
pam, baclofen, and other GABA-enhancing drugs.
8.2.7 Novel, Promising, or Ongoing Applications
8.2.7.1 Alzheimer Disease (AD)

In AD the amyloid-β (Aβ) peptide, derived from amyloid precursor protein, is
viewed as a major pathogenic element in the formation of plaques in the patients’
brains. Naturally occurring antibodies against Aβ have been detected in the serum
and CSF of healthy subjects, and IVIG has been shown to contain autoantibodies
against Aß, suggesting that it may have a therapeutic role via an immune-mediated
Aβ-degrading pathway. In an open-label pilot study of 8 patients, IVIG after
6 months of therapy, increased the level of Aβ peptide in the serum, decreased its
levels in the CSF, and resulted in improved cognitive function prompting a phase II
placebo-controlled study. In 24 AD patients with mild-to-moderate disease IVIG
infusions, either 0.4 or 0.8 g/kg/month after 6 months of therapy, significantly
increased the anti-Aβ antibody and Aβ 40/42 peptides in plasma and decreased Aβ
40/42 peptides in the CSF resulting in significant stabilization of cerebral glucose
uptake measured by PET-18-fluorodeoxyglucose scanning (Dodel et al. 2004).
These results prompted a large phase III trial that randomized 390 patients for
18 months. The results, which were just published, showed that IVIg was not effec-
tive (Relkin et al. 2017).
8.2.7.2 Post-polio Syndrome

This is a chronic degenerative condition clinically characterized by new muscular
weakness, fatigue, and pain that develop many years after an initial attack of acute
paralytic poliomyelitis. Although it is thought to be due to attrition of the surviv-
ing motor neurons (Dalakas 1986), immune activation has been observed consist-
ing of lymphocytic infiltrates in the patient’s spinal cords and even the patients’
muscles (Dalakas 1988) observed even 30 years after the original infection, and
upregulation of RNA for tumor necrosis factor (TNF), IFN-γ, interleukin (IL)-4,
and IL-10 cytokines in the CSF, suggesting the possibility of a persistent smolder-
ing inflammatory response. Following IVIG treatment, IFN-γ and TNF mRNA
levels were reduced in the CSF prompting a controlled trial performed in 135
patients. The results, although underwhelming and of uncertain clinical impor-
tance, did show certain significant differences in some physical activity and qual-
ity of life scores (Gonzalez et al. 2006). These results prompted a phase III
FDA-approved international clinical trial that is currently ongoing.
8.2.7.3 Subcutaneous IgG and Therapeutic Perspectives

in Neurology
Subcutaneous IgG, not for replacement therapy but for immunomodulation, is
becoming attractive as immunotherapy in several autoimmune neurological dis-
eases. After the efficacy of subcutaneous IgG has been documented in uncontrolled
studies in CIDP, MMN, and dermatomyositis, a number of multicenter,
108 M.C. Dalakas
FDA-approved trials are currently ongoing to establish efficacy of IgG as chronic

therapy given subcutaneously in lieu of the intravenous route.
8.2.7.4 Surrogate Biomarkers of Efficacy

As with other immunomodulatory agents, a subset of the neurological patients does
not benefit from IVIG therapy, and, at present, we are unable to predict which are
those patients more likely to respond to IVIg. Although surrogate parameters that
predict response to therapy from the outset are needed, they have not yet been fully
developed or validated. The potential role of molecules such as FcγRIIB and
sialylated Fc and changes in regulatory genes have been explored, but the results,
based on small series, are not adequate to reliably identify those neurological patient
subsets that may be resistant or not adequately responding to IVIg.
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Use of Intravenous Immunoglobulin
in Dermatology 9
Jochen H.O. Hoffmann and Alexander H. Enk
9.1 Expert Review and Update in Dermatology
Good clinical evidence on the effective use of intravenous immunoglobulin (IVIg)

in dermatological conditions can be found as early as 1984 and 1986, when Furusho
et al. (1984) and Newburger et al. (1986), respectively, reported the successful
application of IVIg in Kawasaki syndrome. Seven years later, Dalakas et al. (1993)
reported the first successful randomized controlled trial of IVIg in the treatment of
dermatomyositis. Ever since, IVIg has evolved as an important second- and third-
line treatment option for severe dermatological conditions like autoimmune blister-
ing diseases and scleromyxedema. Other dermatological conditions that may
respond to IVIg treatment include vasculitis and toxic epidermal necrolysis
(Table 9.1). Still, IVIg treatment is off-label for most dermatological indications.
The most current guidelines on the use of IVIg in dermatology were provided by the
European Dermatology Forum in 2016 (Enk et al. 2016). Other comprehensive
guidelines on the use of IVIg exist for the United Kingdom (Provan et al. 2008) and
Australia (Group NICRW 2012).
9.2 Autoimmune Blistering Diseases
Autoimmune blistering diseases encompass dermatological conditions in which the

immune system launches a mainly humoral immune response against self-antigens
involved in keratinocyte cell-cell adhesion (pemphigus group) or the connection
J.H.O. Hoffmann (*) • A.H. Enk

Department of Dermatology, University of Heidelberg, Heidelberg, Germany

112 J.H.O. Hoffmann and A.H. Enk
Table 9.1 Dermatological indications for the use of IVIg

Condition Line of treatment Level of evidence
Pemphigus vulgaris and foliaceus Second or third RCT (Amagai et al. 2009), CS, CR
Bullous pemphigoid Third RCT (Amagai et al. 2017), CS, CR
Mucous membrane pemphigoid, Second or third CS, CR
epidermolysis bullosa acquisita
Dermatomyositis Second or third RCT (Dalakas et al. 1993), CS, CR
Systemic lupus erythematosus Selected cases RCT (Boletis et al. 1999; Perricone
et al. 2008), CS, CR
Scleromyxedema First or second CS, CR
Kawasaki syndrome First CS, CR
ANCA-associated vasculitis Third RCT (Jayne et al. 2000), CS, CR
Toxic epidermal necrolysis Firsta CS, CR
RCT randomized controlled trial, CR case report, CS case series
a
This indication is highly controversial
a b
Fig. 9.1 Pemphigus vulgaris. Images of the chest area of a patient with severe mucocutaneous
pemphigus vulgaris resistant to treatment with oral prednisolone (up to 3 mg/kg body weight),
mycophenolate mofetil (3 g/day), and hemolysis in response to dapsone (a). We initiated concomi-
tant treatment with IVIg (2 g/kg body weight) over a course of 2 days every 4 weeks. Under the
combined immunosuppression, disease activity finally subsided. The dosage of prednisolone and
mycophenolate mofetil could be reduced stepwise. The patient is still in remission after 4 years (b)
between epidermis and dermis (pemphigoid group, epidermolysis bullosa acquis-

ita). The resultant clinical picture is dominated by blisters (especially pemphigoid
group, epidermolysis bullosa acquisita) and erosions (especially pemphigus group)
on the skin and adjacent mucous membranes. Before the advent of immunosuppres-
sive therapy, autoimmune blistering diseases often took a lethal course due to
cachexia and superinfection.
Very convincing clinical evidence from two randomized controlled trials exists
for the use of IVIg in pemphigus vulgaris, pemphigus foliaceus, and bullous pem-
phigoid (Amagai et al. 2017, 2009). Figure 9.1 shows images of a patient with
severe pemphigus vulgaris of the skin and mucous membranes who responded to
9 Use of Intravenous Immunoglobulin in Dermatology 113
Table 9.2 IVIg in autoimmune bullous disease

Indication Second or third line, in combination with systemic steroids and steroid-sparing
immunosuppressants
Dosage 2 g/kg body weight over a period of 2–5 days
Intervals 4 weeks
IVIg treatment. Several case series highlight the effectiveness of IVIg in mucous
membrane pemphigoid and epidermolysis bullosa acquisita. Furthermore, based on
analogy and case reports, the use of IVIg is advocated in severe and treatment-
resistant cases of other autoimmune blistering diseases with a similar pathophysiol-
ogy. IVIg is recommended as an adjuvant second- or third-line treatment after
unsuccessful application of a conventional immunosuppressive combination ther-
apy including a systemic steroid and, most commonly, mycophenolate mofetil or
azathioprine (Eming et al. 2015; Enk et al. 2016; Feliciani et al. 2015; Venning et al.
2012). The initiation of IVIg is usually flanked with high-dose oral corticosteroids
(prednisolone 1–2 mg/kg body weight) to initiate remission. Usually, 2 g/kg body
weight of IVIg distributed over the course of 2–5 days is infused every 4 weeks
(Table 9.2). If no sustained response is observed after four to six treatment cycles,
temporary escalation with the anti-CD20 antibody rituximab can be considered
(Ahmed et al. 2006). In case of a response, the systemic steroid is tapered to 5 mg/
day prednisolone, followed by a taper and discontinuation of the steroid sparing
conventional immunosuppressive. Subsequently, the intervals of IVIg treatment are
extended to 6 weeks, and, finally, IVIg treatment is discontinued.
9.3 Dermatomyositis
Dermatomyositis is an idiopathic inflammatory myositis and a member of the ill-

defined group of collagen vascular diseases. Complement-mediated small vessel
damage is thought to contribute to disease pathogenesis; however, no single patho-
genic model is ubiquitously accepted to date. Apart from mostly proximal muscle
weakness, dermatomyositis presents with photo-distributed erythema and poikilo-
derma, “Gottron” papules over the extensor aspects of the knuckles, and, usually,
prominent alterations of the nail fold capillaries. Due to the involvement of the heart
and lung, and the occurrence of dermatomyositis as a paraneoplastic syndrome, the
mortality in adult patients is considerably high with reported 5-year survival rates
down to 65%. Overlap syndromes, in particular with scleroderma, are not
uncommon.
Systemic steroids are the mainstay of dermatomyositis treatment. The beneficial
effect of adjuvant IVIg on dermatomyositis, in particular on neuromuscular symptoms,
was convincingly demonstrated in a randomized controlled trial (Dalakas et al. 1993)
and multiple case series and reports. The current European IVIg guideline recommends
the addition of IVIg after failure of systemic steroid monotherapy (Enk et al. 2016). The
introduction of a steroid-sparing immunosuppressant, most commonly azathioprine or
Table 9.3 IVIg in dermatomyositis

Indication Second or third line, in combination with systemic steroids; 1st line in severe
cases in combination with systemic steroids
Dosage 2 g/kg body weight over a period of 2–5 days
Intervals 4 weeks
mycophenolate mofetil, can be regarded as an alternative second-line approach

(Sunderkotter et al. 2016). Simultaneous first-line application of systemic steroids and
IVIg may be justified in severe cases (Enk et al. 2016). Usually, systemic steroids are
recommended at high initial doses (prednisolone 1–2 mg/kg body weight). The recom-
mended dosage of IVIg is 2 g/kg body weight over a period of 2–5 days. Treatment
intervals are commonly 4 weeks (Table 9.3). The authors tend to flank initiation of IVIg
treatment with high-dose oral corticosteroids. Response to treatment is usually fast, and
effects can be expected after the third to fourth treatment cycle at the latest. If patients
respond to treatment, systemic steroids can be slowly tapered to 5 mg/day. Subsequently,
IVIg intervals can be extended to 6 weeks, and, finally, IVIg treatment can be
discontinued.
9.4 Systemic Lupus Erythematosus
Systemic lupus erythematosus (SLE) is a protean multisystem autoimmune disease,

which is prominently associated with the production of antinuclear, in particular,
anti-double-stranded DNA autoantibodies. Specific cutaneous manifestations range
from the idiosyncratic butterfly rash to UV-induced hyperkeratotic plaques with
atrophic scarring. Systemic manifestations are manifold and potentially life-
threatening, including lupus nephritis, encephalitis, and arthritis.
Randomized controlled trials highlight the use of IVIg in lupus nephritis, where it
was shown to be equally effective as cyclophosphamide over a course of 18 months
(Boletis et al. 1999), and in pregnant patients with SLE and recurrent spontaneous
abortions, where IVIg reduced lupus activity scores and pregnancy loss (Perricone
et al. 2008). However, the duration of the former study was criticized, as differences
in the efficiency of treatments may not manifest for up to 5 years in SLE (Mulhearn
and Bruce 2015). Several small case series and reports document beneficial effects of
IVIg treatment in a broad range of SLE manifestations. Depending on the involved
organs, current treatment recommendations include systemic corticosteroids, antima-
larials, non-steroidal anti-inflammatory drugs, methotrexate, azathioprine, mycophe-
nolate mofetil, cyclophosphamide, and rituximab (Hahn et al. 2012; Tunnicliffe et al.
2015). Based on the available data, IVIg may be considered as maintenance treatment
in selected cases of lupus nephritis refractory to more established treatment options.
Other manifestations of SLE that may prompt consideration of IVIg include neuro-
psychiatric lupus, in particular, Guillain-Barré syndrome associated with SLE, lupus-
associated immune thrombocytopenia, and SLE in pregnancy (Mulhearn and Bruce
2015). In SLE, IVIg is usually administered at doses ranging from 400 mg to 2 g/kg
body weight over 2–5 days in 4-week intervals (Table 9.4).
Table 9.4 IVIg in systemic lupus erythematosus

Indication Selected cases if other established treatment options failed or are contraindicated
Dosage 400 mg to 2 g/kg body weight over a period of 2–5 days
Intervals Usually 4 weeks
Table 9.5 IVIg in scleromyxedema Indication First or second line

Dosage 2 g/kg body weight over a
period of 2–5 days
Intervals 4 weeks
9.5 Scleromyxedema
Scleromyxedema is a chronic disease of unknown etiology that results in the dermal

deposition of mucin and dermal fibrosis. It is usually associated with a monoclonal
gammopathy. Clinically, patients develop symmetric waxy papules and indurated
plaques mainly on the trunk, face, and upper extremities. Apart from disfigurement,
dermatogenic contractures can severely impair mobility. Internal organ and in par-
ticular neurological involvements are feared and potentially lethal complications.
Successful application of IVIg in scleromyxedema was reported in several case
series and reports. Furthermore, given the frequent association of scleromyxedema
with paraproteinemia, there is a pathophysiologic rationale for beneficial effects of
IVIg. Due to the low effectiveness and potential side effects of other therapeutic
strategies, e.g., chemotherapeutic agents, the current European IVIg guideline sup-
ports the use of IVIg first line in severe cases (Enk et al. 2016). It is recommended
at a dosage of 2 g/kg body weight over a period of 2–5 days in 4-week intervals
(Table 9.5). If the treatment is not effective after 6 months, it should be discontin-
ued. If the patient responds, the treatment intervals can be extended to 6 weeks, and,
finally, treatment can be discontinued. However, relapses after treatment cessation
are not unusual, and long-term treatment may be necessary.
9.6 Kawasaki Disease
Kawasaki disease designates an acute generalized vasculitis that mainly affects chil-
dren. It was hypothesized that the disease might be triggered by an unknown infec-
tious agent in genetically predisposed individuals. Clinically, Kawasaki disease
starts with high fever (>40 °C), which is accompanied by unilateral cervical lymph-
adenopathy in most cases. Subsequently, a pronounced erythema of the tongue and
oral cavity, conjunctival injection, and morbilliform, scarlatiniform, or purpuric
exanthema usually ensue. The disease can be complicated by myocardial, coronary,
neurological, and gastrointestinal involvements, which are mainly responsible for
mortality and residual functional impairment.
Table 9.6 IVIg in Kawasaki Indication First line in combination with

disease acetylsalicylic acid
Dosage 2 g/kg body weight over 12 h
Intervals Single cycle, may be repeated if
standard treatment fails
The mainstay of treatment of Kawasaki disease is the application of 2 g/kg body

weight IVIg over 12 h in combination with acetylsalicylic acid (Table 9.6). If the
standard treatment fails, a second dose of IVIg is usually recommended (Neudorf
and Lilienthal 2013; Newburger et al. 2004). Kawasaki disease is currently the only
dermatological condition for which IVIg use is officially approved by the European
drug agency and US food and drug administration.
9.7 ANCA-Associated Vasculitis
The group of anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis

encompasses granulomatosis with polyangiitis (GPA, Wegener’s disease), eosino-
philic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome), and
microscopic polyangiitis (MPA). Pathophysiologically, ANCA antibodies are
thought to cause neutrophil granulocyte degranulation within small-to-medium size
vessels and subsequent organ damage. Forty to fifty percent of patients experience
skin symptoms, mainly inflammatory nodules, palpable and retiform purpura, and
ulceration, at some point during their disease course. Organ involvement includes
potentially fatal cardiac, pulmonary, and renal damage.
A randomized controlled trial conducted in the year 2000 found a beneficial
effect of adjuvant IVIg in ANCA-associated vasculitis (Jayne et al. 2000). However,
only a single dose of IVIg was used, and the observation period was short (3 months).
Apart from this study, several smaller case series document favorable results under
IVIg treatment. Still, a recent Cochrane review concluded that insufficient evidence
exists for the use of IVIg in GPA (Fortin et al. 2013). According to the current
European IVIg guideline, IVIg remains a valid adjuvant treatment option for
selected refractory vasculitis patients if other, more established treatments failed or
are contraindicated (Enk et al. 2016). IVIg was used at 2 g/kg body weight over a
period of 2–5 days in most published studies. Given the experience in other disease
entities, 4-week intervals can be suggested (Table 9.7).
9.8 Toxic Epidermal Necrolysis
Toxic epidermal necrolysis (TEN), formerly known as Lyell syndrome, is a severe

blistering drug reaction that involves the influx of cytotoxic T-cells into the epider-
mis and a full-blown epidermal necrosis. By definition, patients with TEN present
with skin detachment of at least 30% of the body surface. The mucous membranes
are usually involved. The mortality of TEN ranges from 25 to 70%, depending on
the extent of the disease.
Table 9.7 IVIg in ANCA- Indication Third line, as adjuvant treatment

associated vasculitis Dosage 2 g/kg body weight over a period of
2–5 days
Intervals 4 weeks
Table 9.8 IVIg in toxic Indication Controversial

epidermal necrolysis Dosage ≥3 g/kg body weight over a
period of 2–5 days
Intervals Single cycle
The mainstay of the treatment of TEN is the cessation of the causative drug and
best supportive care. Given the large-scale erosions, patients are usually admitted to
a burn intensive care unit. IVIg contains inhibitory antibodies, in particular, anti-
FAS antibodies that may, in theory, prevent keratinocyte apoptosis. The available
data on the effectiveness of IVIg in TEN is, however, contradictory. Some authori-
ties suggest that IVIg may only be beneficial at high doses in TEN. Indeed, a recent
meta-analysis found an inverse relation between the dose of IVIg and mortality
(Barron et al. 2015). The recommended dosage is, therefore, a single cycle of at
least 3 g/kg body weight IVIg over the course of 2–5 days (Table 9.8). Given the
favorable risk profile of IVIg, the harsh prognosis of TEN, and the lack of reliable
alternative therapeutics, the current European IVIg guideline states that IVIg treat-
ment is justified in TEN (Enk et al. 2016). If the decision for the application of IVIg
is made, however, treatment should commence as early as possible. It is important
to stress that, apart from the cessation of the offending drug and best supportive
care, no generally accepted treatment guidelines for TEN exist. Alternative treat-
ment options that were very recently labeled promising in a comprehensive meta-
analysis by the group of Maja Mockenhaupt (Zimmermann et al. 2017) are systemic
steroids and cyclosporine, while insufficient evidence was found to support the use
of IVIg in TEN.
References
Ahmed AR, Spigelman Z, Cavacini LA, et al. Treatment of pemphigus vulgaris with rituximab and
intravenous immune globulin. N Engl J Med. 2006;355:1772–9.
Amagai M, Ikeda S, Shimizu H, et al. A randomized double-blind trial of intravenous immuno-
globulin for pemphigus. J Am Acad Dermatol. 2009;60:595–603.
Amagai M, Ikeda S, Hashimoto T, et al. A randomized double-blind trial of intravenous immuno-
globulin for bullous pemphigoid. J Dermatol Sci. 2017;85:77–84.
Barron SJ, Del Vecchio MT, Aronoff SC. Intravenous immunoglobulin in the treatment of Stevens-
Johnson syndrome and toxic epidermal necrolysis: a meta-analysis with meta-regression of
observational studies. Int J Dermatol. 2015;54:108–15.
Boletis JN, Ioannidis JP, Boki KA, et al. Intravenous immunoglobulin compared with cyclophos-
phamide for proliferative lupus nephritis. Lancet. 1999;354:569–70.
Dalakas MC, Illa I, Dambrosia JM, et al. A controlled trial of high-dose intravenous immune
globulin infusions as treatment for dermatomyositis. N Engl J Med. 1993;329:1993–2000.
Eming R, Sticherling M, Hofmann SC, et al. S2k guidelines for the treatment of pemphigus vul-
garis/foliaceus and bullous pemphigoid. J Dtsch Dermatol Ges. 2015;13:833–44.
Enk AH, Hadaschik EN, Eming R, et al. European Guidelines (S1) on the use of high-dose intra-
venous immunoglobulin in dermatology. J Eur Acad Dermatol Venereol. 2016;30:1657–69.
Feliciani C, Joly P, Jonkman MF, et al. Management of bullous pemphigoid: the European
Dermatology Forum consensus in collaboration with the European Academy of Dermatology
and Venereology. Br J Dermatol. 2015;172:867–77.
Fortin PM, Tejani AM, Bassett K, et al. Intravenous immunoglobulin as adjuvant therapy for
Wegener’s granulomatosis. Cochrane Database Syst Rev. 2013; CD007057.
Furusho K, Kamiya T, Nakano H, et al. High-dose intravenous gammaglobulin for Kawasaki dis-
ease. Lancet. 1984;2:1055–8.
Group NICRW. Criteria for the clinical use of intravenous immunoglobulin in Australia. 2nd ed.
In: 2012.
Hahn BH, Mcmahon MA, Wilkinson A, et al. American College of Rheumatology guidelines for
screening, treatment, and management of lupus nephritis. Arthritis Care Res. 2012;64:797–808.
Jayne DR, Chapel H, Adu D, et al. Intravenous immunoglobulin for ANCA-associated systemic
vasculitis with persistent disease activity. QJM. 2000;93:433–9.
Mulhearn B, Bruce IN. Indications for IVIG in rheumatic diseases. Rheumatology (Oxford).
2015;54:383–91.
Neudorf U, Lilienthal E. Vaskulitiden – Kawasaki-Syndrom. In: AWMF – Leitlinien. 2013.
Newburger JW, Takahashi M, Burns JC, et al. The treatment of Kawasaki syndrome with intrave-
nous gamma globulin. N Engl J Med. 1986;315:341–7.
Newburger JW, Takahashi M, Gerber MA, et al. Diagnosis, treatment, and long-term management
of Kawasaki disease: a statement for health professionals from the Committee on Rheumatic
Fever, Endocarditis and Kawasaki Disease, Council on Cardiovascular Disease in the Young,
American Heart Association. Circulation. 2004;110:2747–71.
Perricone R, De Carolis C, Kroegler B, et al. Intravenous immunoglobulin therapy in pregnant
patients affected with systemic lupus erythematosus and recurrent spontaneous abortion.
Rheumatology (Oxford). 2008;47:646–51.
Provan D, Chapel HM, Sewell WA, et al. Prescribing intravenous immunoglobulin: summary of
Department of Health guidelines. BMJ. 2008;337:a1831.
Sunderkotter C, Nast A, Worm M, et al. Guidelines on dermatomyositis—excerpt from the inter-
disciplinary S2k guidelines on myositis syndromes by the German Society of Neurology. J
Dtsch Dermatol Ges. 2016;14:321–38.
Tunnicliffe DJ, Singh-Grewal D, Kim S, et al. Diagnosis, monitoring, and treatment of systemic
lupus erythematosus: a systematic review of clinical practice guidelines. Arthritis Care Res.
2015;67:1440–52.
Venning VA, Taghipour K, Mohd Mustapa MF, et al. British Association of Dermatologists’ guide-
lines for the management of bullous pemphigoid 2012. Br J Dermatol. 2012;167:1200–14.
Zimmermann S, Sekula P, Venhoff M, et al. Systemic Immunomodulating therapies for Stevens-
Johnson syndrome and toxic epidermal necrolysis: a systematic review and meta-analysis.
JAMA Dermatol. 2017;153(6):514–22. https://doi.org/10.1001/jamadermatol.2016.5668.
Part II
Basics of IgG Concentrates
Historical Aspects of Polyclonal IgG
Preparations 10
Volker Wahn and Peter Späth
10.1 Introduction
Today we can choose between several polyclonal IgG products for both replace-
ment and immunomodulation. However, it was a long way to go to reach this stage.
In this chapter, we try to illustrate the major stages of IgG product development
which began more than 70 years ago.
10.2 Development of Standard IgG
The development of plasma fractionation was a WW II effort with a primary aim to

provide human albumin for battlefield injuries. The technique was developed in
Boston under the lead of Edwin Joseph Cohn (1892–1953) and was made possible
through the strong support of the US Department of Navy, the Office of Scientific
Research and Development, and the wartime blood donor program of the American
Red Cross (Cohn et al. 1944). Gamma globulin was enriched at high purity in frac-
tion II of the Cohn-Oncley cold ethanol fractionation method (Oncley et al. 1949).
This “standard IgG” at increasing amounts became available from 1943 onward, a
time point when the fractionation of albumin has been transferred to industry
(Armour Pharma at Kankakee, IL, USA). Indeed, between 1944 and 1948, approxi-
mately 1 mio doses of “standard IgG” were applied in the USA.
V. Wahn (*)
P. Späth
Institute of Pharmacology, University of Bern, Bern, Switzerland

122 V. Wahn and P. Späth
10.3 T
he Early Days of Clinical Development
of IgG Therapies
From 1941 onward, therapies with protein concentrates from fractioned plasma were
developed under contract between the Office of Scientific Research and Development
and Boston Harvard University. Charles A. Janeway (1909–1981) was put in charge.
At that time, he was running the infectious and immunology laboratory at the Peter
Bent Brigham Hospital and was member of the Harvard Medical School Department
of Bacteriology and Immunology, and in 1940 he furthermore joined the laboratory
of Edwin J. Cohn in the Harvard Medical School Department of Physical Chemistry
(Geha 2005; Rosen and Janeway 1994). After initial studies in 1941 in humans with
bovine (fatal outcomes) and human serum albumin (successful), in 1943 he turned to
study Cohn fraction II (+III), the plasma fraction(s) enriched in IgG. The initial stud-
ies largely remained restricted to the prevention or attenuation of viral infections,
particularly of measles infections (Ordman et al. 1944). The initial restriction to viral
diseases probably was because from the late 1930s onward, the dawn of antibiotic
treatment, Janeway built up an outstanding expertise in the treatment of bacterial
infections with these emerging new drugs (Smith 1977).
The first human ever receiving an IgG concentrate was Janeway himself—with
almost fatal consequences as the lot was contaminated with bacterial toxin (Rosen and
Janeway 1994). Aiming for rapid increase of antibodies in the circulation and pursu-
ing the “tradition” set, the “lot-release” for intravenous application of toxin free prep-
arations was a self-infusion to one or the other collaborators of Janeway. Repeated
severe and one almost fatal adverse event let Janeway note: “One mystery about nor-
mal serum gamma globulin which has defied explanation is its toxicity on intravenous
injection. Although reactions have been practically nonexistent on intramuscular
injection for measles prophylaxis, intravenous injection of the most highly purified
preparations into normals in moderate doses and into patients ill with acute infections
in much smaller doses has led to acute vasomotor reactions followed by severe chills
and hyperpyrexia. This has occurred so regularly that one wonders whether it has
genuine physiologic significance” (cited from Janeway 1948). Therefore, intravenous
application of “standard IgG” was given up in favor of the intramuscular route.
10.4 F
rom Intramuscular “Standard IgG”
to Intravenous Preparations
Until the early 1950s, two slightly different cold ethanol fractionation methods were
established. In the USA the Cohn-Oncley method provided a highly pure “standard
IgG.” The method was a lavish one with high volumes during fractionation and a
low recovery of IgG (Cohn et al. 1944; Oncley et al. 1949). The other was the
Kistler-Nitschmann method established as the “European” method for plasma frac-
tionation (Nitschmann et al. 1954). With this method, volumes to handle during
fractionation were considerable lower and the recovery higher at cost of some impu-
rities, mainly IgA. Both methods provided a “standard IgG” concentrate.
10 Historical Aspects of Polyclonal IgG Preparations 123
Most likely gamma globulin therapy would have stayed in the shadow of antibi-
otic treatment, if not agammaglobulinemia would have been described in 1952 by
OC Bruton. Applying plasma electrophoresis to the serum of an 8-year-old boy, he
realized an association between recurrent severe infections (starting at an age of
four and a half years of age) and a very low gamma globulin in serum. In an attempt
to reduce susceptibility to infections, he administered “standard IgG” subcutane-
ously and demonstrated an increase of gamma globulin in blood, clinically a
decrease of infections. Some remarkable facts of Bruton’s work have to be high-
lighted: (a) despite the overwhelming position of the Boston group, he selected the
less painful subcutaneous route of administration for his young patient; (b) he
detected a new disease; and (c) he provided the therapy. Although less painful, at
that time, only low doses of “standard IgG” could be administered via this route,
and the need for i.v. preparations allowing administration of larger doses became
evident (again).
10.5 Initial Problems with IVIG Administration
The first human immunoglobulin preparations were Cohn fraction II at 16% strength
without any dedicated polishing step, i.e., a “crude” IgG concentrate. The effort of
Barandun and colleagues shed light on Janeways “mystery about normal serum
gamma globulin”: “standard IgG” contained IgG aggregates capable of activating
the complement system. This activation process generated anaphylatoxins like C3a
and C5a and caused acute intolerance reactions if “standard IgG” was given intrave-
nously. Thus, from the beginning of the 1960s onward, research focused on the
reduction of aggregates as the cause of anticomplementary activity (ACA) in order
to create preparations that were tolerated upon intravenous use. Even today, ACA
assessment is a release criterion for IVIG lots.
10.6 Chemical Procedures to Improve Tolerance
Within this chapter, it is impossible to discuss all details of industrial procedures

developed in order to make human immunoglobulins applicable by i.v. the route.
Thus, only some principles of polishing “standard IgG” are summarized (historical
examples mostly with modification of the molecules):
–– Harsh pepsin digestion leading to 5S F(ab’)2 fragments devoid of Fc-effector

functions and shortened half-life in vivo (Schultze and Schwick 1962)
–– Plasmin digestion (Sgouris 1967)
–– Limited sulfitolysis (Masuho et al. 1976)
–– Reduction and alkylation (Wright 1978)
–– Anion exchange chromatography and PEG precipitation
–– Treatment with ß-propiolactone (Stephan 1969), an IgM/IgA enriched product
remained on the market in some countries
Only a very few of these products are still available in some regions of the world,
mainly because the above measures to achieve i.v. tolerability provided markedly
impaired structures and functions of IgG. Some procedures destroyed IgG3.
Currently, the plasma fractionating companies use combinations of procedures to
achieve high recovery as well as high purity and leave their product as native as pos-
sible, nevertheless well-tolerated by the i.v. route (see Chaps. 12 and 13).
10.7 The First Conference on IVIG Quality
In pivotal trials, rarely placebo controlled, the biological activity of most of the
older products was assessed measuring protection from infections of antibody-
deficient patients. Head-to-head comparisons of different products in vivo or
placebo-controlled trials were rarely performed. A few clinical trials in patients with
primary Immunodeficiencies illustrating developmental steps should be mentioned:
Ammann et al. 1982; Steele et al. 1987; Garbett et al. 1989; Schiff et al. 1997;
Lamari et al. 2000; Roifman et al. 2003; Kallenberg 2007).
In order to define some quality characteristics for IgG preparations a WHO/IUIS
expert committee met in 1983 and defined minimal requirements for IVIG products
(IUIS 1983):
–– Obtained from plasma pools from at least 1000 donors

–– Prekallikrein activator (PKA) activity below a predefined threshold level
–– Kinins below a predefined threshold level
–– Anticomplementary activity (ACA) below a predefined threshold level (a general
lot release criterion)
–– Plasmin content below a predefined threshold level
–– No accumulative preservatives (i.e., Merthiolate)
–– IgG content at least 90% (monomers + dimers, low aggregate content)
–– IgG as native as possible, i.e., chemically unmodified, retained biological func-
tions such as antigen recognition and Fc functions
–– Physiological IgG subclass distribution
–– Titer of some selected specific antibodies guaranteed, a lot release criterion vary-
ing from brand to brand
–– Low IgA, absence or minute amounts of IgE
–– Isohemagglutinins below a predefined threshold level
–– Alloantibodies (i.e., anti-D and others) below a predefined threshold level
At that time, not all products fulfilled these requirements. However, keeping
these goals in mind the different companies intensified their research heading for
products that came as close to these requirements as possible. After a one and half
decade of development, the first “native” 7S IgG concentrate came to the market in
Switzerland: Ig-SRC (SRC = Swiss Red Cross). The clue to reach intravenous toler-
ability was the polishing of “standard IgG” at low pH and traces of pepsin. This step
rendered remaining aggregates ineffective. Other manufacturers applied other
techniques to get rid of IgG aggregates or to render them non-complement activat-

ing. Much progress in polishing steps was made leading to different products with
lyophilized or liquid formulation needing different stabilizers (Cherin et al. 2016).
10.8 Adverse Events
Severe adverse events (sAEs) from the very beginning of transfusion science and its
clinical application have been a threat to the recipients. sAEs encompass incompat-
ible transfusion reactions, TRALI, anaphylactic reactions, organ damage, and trans-
mission of pathogens. Plasma products mediate some of these sAEs as well. Back
in the early 1940s, an elevated risk for “homologous serum jaundice” was associ-
ated with the use of pooled serum (for stabilizing yellow fever vaccine). The same
was true for the first product prepared from pooled plasma, albumin (Spurling et al.
1946). Great efforts made already in 1945 available a virus inactivation method for
albumin concentrates, pasteurization at 60 °C for 10 h (Gelli et al. 1948).
Unfortunately, this method was not applicable to “standard IgG” and sporadic trans-
mission of hepatitis occurred (see below).
The recommendations by the WHO/IUIS expert committee in 1983 were based
on causes of adverse events known at that time. A major problem at that time were
immediate anaphylactoid reactions caused by anti-IgA antibodies (IgG or IgE iso-
type) in the patients and “phlogistic” reactions delayed by about 2–3 h after start of
the infusion (= patient-related) or anticomplementary, PKA or kinin activity in the
products (= product-related).
10.8.1 Pathogen Transmission
The worst of the adverse events with IgG concentrates which occurred after the
WHO/IUIS recommendation was the transmission of hepatitis C virus (HCV; “hep-
atitis non-A, non-B” termed at that time) by some polyvalent IgG preparations
(Lane 1983; Lever et al. 1984; Stephan and Dichtelmüller 1983; Weiland et al.
1986; Welch et al. 1983; Williams et al. 1989) as well as with an anti-D immuno-
globulin prepared in the former German Democratic Republic. It became apparent
that Cohn fractionation II of human plasma alone was limited in its capacity to
inactivate transmissible viruses while products remained free from the threat of
virus transmission, particularly those prepared by the Kistler-Nitschmann fraction-
ation technique, most likely due to the low pH applied at polishing. Several patients
infected by HCV experienced a severe course of infection and a few died (Björkander
et al. 1988; Razvi et al. 2001) making viral safety a crucial issue for IgG products
(Cuthbertson et al. 1987). Driven by the virus transmission risks of blood transfu-
sion and the obvious elevated risk of virus transmission by coagulation factor con-
centrates, authorities always orient themselves to the highest risk level and require
accordingly measures to guarantee product safety. IgG therapy on one hand com-
prises a particular set of risks, while on the other hand the fractionation technique
and the polishing steps offer particular opportunities for virus inactivation/removal.
The particular risks with IgG therapy are:
–– Economic reasons force manufacturers to manipulate at once large volumes of

pooled plasma.
–– One contaminated plasma unit can contaminate thus a large pool.
–– One lot of IgG concentrate has many vials for clinical use.
–– The many vials are applied to a certain (high) number of patients and in case of
contamination infections are usually clustered in a patient population.
–– Recipients might be individuals with genetically impaired antibody production.
Such patients are particularly vulnerable to infections. Such patients receive IgG
concentrates for replacement therapy possibly lifelong, and patients are pro-
longed and repeatedly exposed to (a theoretical) risk of pathogen transmission.
–– Patients with inherited immunodeficiency might need lifelong replacement ther-
apy which exposes them to repeated risk of virus transmission.
–– Patients with chronic autoimmune and/or inflammatory diseases are treated with
“immunomodulatory” doses of IgG. The doses are high and usually are applied
repeatedly, and this again exposes patients to an elevated level of risk for patho-
gen transmission (Table 10.1).
Table 10.1 Transfusion medicine from the very early days onward was struggling with transmis-
sion of pathogens, particularly viruses
Genus Viruses
Herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), human
herpesviruses (HHV)-6, -7, -8
Papovaviruses John Cunningham (JC) and BK (initials patients) viruses
Parvoviruses Parvovirus B19V, adeno-associated virus (AAV)
Hepadnaviruses Homologous serum jaundice (HBV)
Circoviruses Transfusion transmitted virus (TTV), TTV-like mini virus or torque teno
mini virus (TLMV)
Retroviruses Human immunodeficiency virus (HIV)-1,-2, human T-cell
lymphotropic virus (HTLV III, LAV)
Flaviviruses Hepatitis C virus (HCV), West Nile virus (WNV), Zika virus (ZIKV),
yellow fever virus (YFV), other arboviruses
Alphaviruses Chikungunya virus (CHIKV), (W,E,V) equine encephalosis viruses
(EEVs)
Coronaviruses SARS-associated virus
Bornaviruses Borna disease virus
Picornaviruses Hepatitis A virus (HAV), human enteroviruses
Bunyaviruses La Crosse, Sin Nombre, Hantaan
Arenaviruses Lassa fever, Junin, Machupo
Hepaviridae Hepatitis E virus (HEV)
Listed are some viruses found in blood donations. Pooling of plasma elevates the risk of infecting
clusters of recipients by a single lot of a plasma product. Those viruses, which might represent a
potential threat for transmission by plasma products, are depicted in bold
In order to lower the risk of pathogen transmission, validated processes for elimi-
nation/inactivation of pathogens became mandatory for plasma products (see
Chap. 12).
It needs to be mentioned that up to date not a single case of HIV infection
acquired through IgG preparations even before dedicated virus inactivation and
removal steps have been introduced to the fractionation and polishing processes.
Furthermore, no case of variant CJD transmission by plasma products has been
reported worldwide (Helbert et al. 2016). The problem with hepatitis C seems to be
solved because since more than 25 years no new cases of HCV transmission have
been reported. Nevertheless all companies producing IgG concentrates are
extremely alert with respect to pathogens possibly emerging/re-emerging in the
future.
10.8.2 Noninfectious Adverse Events
A coevolution of noninfectious adverse event profiles with “improved” products,

routes of application, doses applied, increased infusion rate, and the more broad
therapeutic use of IgG concentrates has occurred (Feldmeyer et al. 2010; Berger
2013; Dantal 2013). Reasons for the adverse effects are multiple (Späth et al. 2015).
Parameters controllable by, e.g., polishing steps or the use of appropriate
stabilizers:
–– Harming of the IgG molecules inherent to any fractionation process.

–– Chemicals to stabilize the concentrates during their shelf life.
–– Inappropriate handling of the concentrates during their shelf life.
–– Alteration of the IgG molecules due to inappropriate handling before infusion
(foam).
–– Presence of too high amounts of preformed dimers in the preparation.
–– Skin reactions at site of infusion/injection.
–– Too rapid increase of exogenous IgG in the circulation, a combination of infu-
sion rate and strength of the solution infused.
–– Increase in recovery might lead to an altered population of IgG molecules result-
ing in an altered adverse event profile.
Parameters not controllable:
–– The immune status of the diseased patient at the time point of the infusion, i.e.,
the subtle and extreme wide array of recognition by infused IgG of the patients’
immune structures and vice versa, the recognition by patients’ immune system,
the exogenous IgG (for details see P. Späth et al. 2015).
Delayed adverse reactions might all be associated with risk factors the diseased
patient brings along. These factors are discussed to render patients particularly sen-
sitive to effects of the interrelation of applied IgG and patient’s immune status. Such
“risk factors” may exist subclinically. Some of the more common adverse events
seen primarily with IVIG are (modified from Cherin et al. 2016):
–– Migraine headaches
–– Aseptic meningitis (patient dehydrated? Local meningeal inflammation induced
by IgG on the basis of infused IgG recognizing “risk factors?”) (Sekul et al.
1994; Scribner et al. 1994; Hopkins and Jolles 2005; Berg and Fuellenhals 2016)
–– Osmotic nephrosis caused by sugars like saccharose in the products, associated
with “risk factors” of the kidney
–– Other renal impairments due to diuretics and renin–angiotensin system
inhibitors
–– Thrombosis/embolic events and myocardial infarction (probably caused by acti-
vated faxtor XI, high sodium content, and high osmolality) (Hefer and Jaloudi
2004; Elkayam et al. 2000; Ammann et al. 2016)
–– Hemolysis (probably caused by isohemagglutinins and alloantibodies)
–– Neutropenia (cause unknown)
–– Transfusion-related acute lung injury (TRALI; speculation on pathogenetic role
of anti-neutrophil antibodies)
–– Hyponatremia, pseudohyponatremia (rare, defect in urinary free water
excretion?)
–– Hyperviscosity syndrome (possibly caused by preexisting very high levels of
IgG in patients) (Hague et al. 1990; Oh et al. 1997)
–– Necrotizing enterocolitis in newborn babies (cause unknown, immaturity?)
(Figueras-Aloy et al. 2010; Kara et al. 2013; Navarro et al. 2009; Yang et al.
2016)
These observations have stimulated further improvements of the products:

Saccharose was replaced by other stabilizers, by validation studies elimination of
potential procoagulant activity (demonstrating activated factor XI) during the man-
ufacturing process has had to be shown by each company; some of these studies
have been published (José et al. 2013; Williams et al. 2013). The rate of hemolytic
adverse events has increased with chromatographically produced IgG concentrates.
Attempts to reduce these rates include screening of individual plasma donations and
withholding from pooling very high isohemagglutinin titer donations and/or polish-
ing by affinity column chromatography steps to lower these titers (Dhainaut et al.
2013; Siani et al. 2014; Gerber et al. 2016). With the expanding use of IgG concen-
trates for subcutaneous application, any adverse events have declined
considerably.
10.9 Summary
Manufacturing and safe clinical use of IgG concentrates has gone a long stony but
finally successful way. Compared to “old” products currently available, IgG con-
centrates combine better recovery and higher purity, are more convenient in their
use due to liquid formulation and storage at room temperature during their shelf life,
and have a higher pathogen safety, tolerability, purity, and efficacy than the products
40 years ago (Gelfand 2006; Cherin et al. 2016).
The development of products for s.c. administration expanded the spectrum of
treatment options. The option of self-administration by patients increased their
quality of life because this treatment can be given at home. S.c. products also help
to treat patients who have repeated intolerance reactions to IVIG. S.c. products are
increasingly studied in chronic autoimmune/inflammatory conditions, e.g., chronic
inflammatory demyelinating polyradiculoneuropathy (CIDP), multifocal motor
neuropathy (MMN), and various dermatological and collagen-vascular diseases.
Results from clinical trials will be available soon. Whether or not ITP can be treated
by s.c. route remains to be clarified. One boy with ataxia telangiectasia developed
ITP while being on s.c. replacement for his immunodeficiency (Heath and Goldman
2010).
The availability of human IgG has enabled clinicians all over the world to treat
several diseases with relatively low toxicity. Nevertheless, the obstacles illustrated
in this chapter should be kept in mind to motivate authorities and the plasma indus-
try to enforce efforts to further optimize their products.
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Basics of Immunoglobulins as Effector
Molecules and Drugs 11
Tchavdar L. Vassilev, Victor Kostov, Stephan von Gunten,
and Anastas D. Pashov
11.1 Introduction
Immunoglobulins (antibodies) are glycoprotein molecules that play a key role in

adaptive immunity. They protect us in the hostile environment of bacteria, viruses,
and parasites. There are five classes of human immunoglobulins—IgM, IgG, IgA,
IgE, and IgD. Most IgM antibodies (1.5 mg/mL plasma) are “natural”—i.e., they
are produced even without an antigenic stimulus. Their antigen-binding polyspeci-
ficity ensures them a role as a first line of defense mechanism against invading
pathogens. The therapeutic potential of pooled IgM has not been utilized in clinical
practice yet. IgG is the most abundant immunoglobulin isotype in human plasma
(12 mg/mL), and this chapter is devoted mostly to it. The ability of IgG antibodies
to bind with a high affinity and specificity to a remarkably large variety of antigens
is their main feature. Serum IgA (3 mg/mL, 90% as monomers, 10% dimeric) has
effects similar to these of IgG, while secretory IgA antibodies are resistant to prote-
ases and protect all mucosal surfaces. IgE is the class with the lowest plasma con-
centration (0.05 mcg/mL). The contact of mast cell-bound IgE with the specific
antigen results in an acute inflammatory reaction that might help to expel parasites
from the gut. IgE antibodies are also believed to have a role in the host defense
against noxious environmental substances, including venoms, environmental xeno-
biotics, and irritants (Palm et al. 2012). The plasma concentration of IgD is highly
variable (mean value 25 mg/mL), and the biological role of antibodies of this iso-
type is poorly understood.
T.L. Vassilev (*)

UniMed Medical Center, Sevlievo, Bulgaria
V. Kostov • A.D. Pashov
Stephan Angelov Institute of Microbiology, Sofia, Bulgaria
S. von Gunten

134 T.L. Vassilev et al.
The future of the use of antibodies as drugs for the prevention and treatment of
infectious, autoimmune diseases and cancer is bright. The global normal pooled
intravenous immunoglobulin (IVIg) market grows by 6.8% each year and is
expected to reach $11.6 billion (230 tons) by 2021 (see http://www.prnewswire.
com/news-releases).
11.2 Structure and Functions of IgG Molecules
11.2.1 The F(ab)2 Fragment
IgG is composed of two heavy and two light polypeptide chains linked to each
other by disulfide bonds. Each of the two antigen-binding (Fab) fragments is com-
posed of a light chain and a portion of a heavy chain. The light chain has two
immunoglobulin domains—regions of compactly folded structure—while the
heavy chain has four (see Fig. 11.1). The amino-terminal domains of both chains
are highly variable (V domains), while the others are referred to as “constant.” The
variable (V) domains of both form the antigen-binding site (paratope). They are not
encoded in full in the genome, but by a combination of multiple variants of differ-
ent gene segments. Two or three gene segments (for the V regions of light and
heavy chains, respectively) are assembled by a process known as gene rearrange-
ment forming a complete V-region sequence. There are 34–38 V-kappa and
5 J-kappa; 29–33 V-lambda and 4–5 J-lambda light chain V segments, as well as
Idiotype
VL
VL
VH CL
CL
VH
Fig. 11.1 The structure of

an IgG molecule. The H
1
C C3b- and C4b-
CH
heavy immunoglobulin
1
Fab binding region

chain and its four domains
are shown in green and the Hinge region
light in red. The inter-chain
disulfide bonds are in blue.
The variable (V) regions of C1q-binding region
CH2
CH2
both chains of the Fab

fragments shape the two Fc
antigen-binding sites carbohydrate
(paratopes) of the antibody.
CH3
CH3
Its idiotypic determinants

(idiotopes) are in the same
area. The biding sites of
complement components
and Fc receptors are also
shown Fcγ receptor types I, II, III
11 Basics of Immunoglobulins as Effector Molecules and Drugs 135
36–46 V-, 23 D-, and 6 J functional heavy chain V-gene segments. The combina-
tion of any V-light chain variant with any V-heavy chain should result in approx.
2.106 antigen-binding site specificities. Further diversity is generated by the sub-
traction (by endonucleases) and by the addition (by the enzyme terminal deoxy-
nucleotidyl transferase (TdT) and by the incorrect DNA chain repair) of nucleotides
during the process of gene fragment recombination. The somatic hypermutation of
the already rearranged V-region genes of the activated B cells in the secondary
lymphoid organs increases the diversity still further. These processes result in the
ability to generate about 1012 different specificities of immunoglobulin B cell
receptors and of circulating immunoglobulins. B cells with strong anti-self reac-
tivities are eliminated, become anergic, or change their receptors by receptor edit-
ing in the bone marrow. The repertoire of immunoglobulin B cell receptors and
circulating immunoglobulins is regarded as being quasi-complete—i.e., although
purged of strong self-reactivities, they can still interact with practically any xeno-
antigen. The same is true for the antibody diversity in pooled therapeutic immuno-
globulins produced from healthy donor plasma pools. The antibody repertoire is
discussed in more detail below.
Between 15 and 25% of all Fab variable regions have additional complex
branched glycans linked to N-glycosylation sites that emerge during somatic hyper-
mutation. These glycans affect antigen binding by both BCR and circulating immu-
noglobulins (van de Bovenkamp et al. 2016).
11.2.2 The Fc Fragment
The Fc fragments are built of a pair of the CH2 and CH3 heavy chain domains
(Fig. 11.1). The two CH3 domains are paired, while the two CH2 are not. Fc frag-
ments are much less variable than the Fab antibody fragments. In addition to iso-
typic and allotypic differences, there are also hundreds of variants of the glycans
coupled to it. The “hinge” region is located in between the F(ab)2 and Fc fragments.
Its length and flexibility determine the ability of both Fab arms to assume different
arm angles—from 00 to 1800. IgG1 and IgG3 antibodies have longer and flexible
hinge regions, and this relates to their strong ability to activate complement and
antibody-dependent cell cytotoxicity. IgG2 and IgG4 have short hinge regions and
are weak complement activators.
The IgG binding to a toxin or to a virus particle can prevent the interaction with
their surface receptors on target cells, thus neutralizing them. For other pathogens,
however binding is not sufficient to disarm them. To fight them the Fc-fragment-
dependent activation of complement and/or Fc receptors on immune cells is needed.
This has as a result the launching of effector molecules—reactive oxygen species,
cytokines, as well as antibody-mediated killing by complement-dependent cytotox-
icity (CDC), by antibody-dependent cell cytotoxicity (ADCC), and by the antibody-
dependent cellular phagocytosis (ADCP).
While monomeric IgG does not bind the C1q complement component, IgG that
is part of immune complexes does so efficiently. Activating the complement system
through its classical pathway results in the complement-mediated killing of the

pathogen. Other component cascade components—C3b and C4b—attach to a site
between the CH1 and VH domains of the heavy immunoglobulin chain and might
interact with the corresponding receptors.
The IgG Fc fragments also allow for an active transport of these molecules to
parts of the body which would have been otherwise unreachable—e.g., the human
fetus.
The glycans attached to asparagine 297 of both heavy chain CH2 domains repre-
sent 3% (by weight) of IgG molecules. Their backbone structure is biantenary and
carries N-acetylglucosamine, mannose, fucose, galactose, and sialic acid and other
residues. The glycans of both heavy chains of an IgG molecule could have identical
or different structures. These structures depend also on the diet and are modified in
patients with autoimmune diseases.
The removal of the Fc-linked glycan suppresses the effector functions of the IgG
molecule. Removal of the N-glycan impairs binding to activating FcR’s and com-
plement activation. The infusion of the bacterial IgG glycan-hydrolyzing enzyme
EndoS has been shown to have the potential to suppress anti-self antibody-mediated
autoimmunity (Hirose et al. 2012). Even minor changes in the glycan structure
result in strong modification of these functions. These modifications could be clini-
cally relevant, e.g., the engineered anti-CD20 antibody obinutuzumab with reduced
fucosylation is used for the treatment of patients with chronic lymphocytic leuke-
mia and follicular lymphoma who have not responded to a fucosylated anti-CD20
antibody (rituximab) (Quast et al. 2017).
11.2.3 Fc-Gamma Receptors
While the antigen-binding specificity of IgG molecules is only F(ab)2 dependent,

most biological effects depend on the ability of the Fc portion to engage various
receptors on immune cells.
The neonatal Fc receptor for IgG (FcRn) stays apart from the other human Fcγ
receptors because of its peculiar structure and functions. Unlike the others its struc-
ture resembles that of a MHC class I molecule. It binds to all IgG subclasses, and this
interaction is not dependent on the glycosylation state of the immunoglobulin mole-
cules. The FcRn receptor is responsible for the transplacental transport of IgG to the
fetus. This receptor binds IgG in acidic vesicles at a pH < 6.5 and releases IgG at a
pH > 7.4 in the blood. The long half-life of these maternal antibodies in the newborn
child ensures a sufficient degree of protection until the start of the production of its
own antibodies. Another important function of FcRn is its role in continuous IgG
recycling which contributes toward the long half-life of immunoglobulins of this
class in the circulation—21 days. In addition FcRn also binds and recirculates
albumin.
The classical type I Fc receptors (FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa,
and FcγRIIIb), being members of the big immunoglobulin superfamily, are present
in unequal combinations on many cells of the immune system (see Table 11.1). In

addition, their expression levels vary on different cell types. FcγRIIb is the only
known inhibitory receptor of this group. This activity is due to the ITIM (immuno-
receptor tyrosine inhibitory motif) on its cytoplasmatic tail. It suppresses signaling
through other stimulatory FcγRs, immunoglobulin receptors on B cells, etc. FcγRIIb
binds weakly monomeric IgG, but interacts with IgG-containing immune com-
plexes. The co-ligation of FcγRIIb and the BCR receptors of B cells results in the
recruitment of SHIP (SH2-containg inositol phosphatase) to the BCR receptor com-
plex, resulting in blocking the PI3-kinase. Blocking the PI3-kinase in turn reduces
the availability of 3-phosphorylated phosphoinositide second messengers, critical
for a variety of signaling pathways including phospholipase C activation and cell
survival.
FcγRI is the only receptor with high affinity to IgG that allows it to bind mono-
meric IgG molecules. The FcγRII and FcγRIII are binding with low-affinity and
high-avidity immune complexes and aggregated IgG, but not monomeric IgG. IgG
complexes are generated during an ongoing immune response to invading patho-
gens and induce various inflammatory reactions by signaling through the above-
mentioned receptors. In a healthy individual, such FcγRII and FcγRIII activation
does not occur, and thus, highly undesirable excessive signaling and inflammatory
reactions are avoided.
CD23 and DC-SIGN belong to the group of nonclassical type II C-lectin recep-
tors. They bind a site between the CH2 and CH3 regions of the Fc fragments.
CD23 has been known as a receptor for IgE, but has recently been show to bind
also to sialylated IgG. The functional consequences of these interactions remain
poorly understood. DC-SIGN binds glycosylated proteins. The group of
F. Nimmerjahn has claimed over the last years that the binding of Fc-sialylated
IgG to DC-SIGN leads to the upregulation of the inhibitory FcγRIIB on macro-
phages and dendritic cells. The authors consider this upregulation as the main
mechanism responsible for the beneficial immunomodulatory effects of immuno-
globulin therapy in autoimmune diseases (Schwab and Nimmerjahn 2013). We
and others have argued that the mechanisms of action of IVIg—a very complex
and pluripotent drug—could hardly be explained by such a simplistic approach
(von Gunten et al. 2014).
Table 11.1 Overview of the cell expression and Fc-binding affinity of human type 1 FcγRs
Type 1 FcγRs
FcγRI FcγRIIa FcγIIb FcγRIIc FcγRIIIa FcγRIIIb
Expression Monocytes, Myeloid B, myeloid, NK cells NK cells, Granulocytes
macrophages, and and dendritic macrophages,
granulocytes dendritic cells monocytes
cells,
platelets
Fc-binding High Low Low Low Low Low
affinity
11.2.4 IgG Subclasses
There are four subclasses of human IgG—IgG1 (approximately 7 mg/mL plasma),

IgG2 (3 mg/mL), IgG3 (1 mg/mL), and IgG4 (0.5 mg/mL). While their amino acid
sequences are 90% identical, they all have different heavy chains, hinge regions,
and differing functional properties: placental transport, antigen binding, immune
complex formation, ability to activate complement, ability to interact with activat-
ing and inhibitory Fc-gamma receptors on cell surfaces, and different half-lives in
the plasma. The production of different IgG subclasses after immunization depends
on the nature of the antigen and the cytokine milieu during the ongoing immune
response. Cases of selective deficiency of each IgG subclass are rare but are infor-
mative about their roles in vivo.
Most protein-binding antibodies belong to the IgG1 subclass. Its decreased lev-
els in primary and secondary immune deficiencies result in frequent infections.
Most of the antibodies to carbohydrate antigens are of the IgG2 isotype. Their pla-
cental transfer and binding to Fc receptors is weak. The consequences of IgG2 defi-
ciency are repeated infections caused by encapsulated bacteria—Streptococci,
Meningococci, etc. The polysaccharide capsule protects these microorganisms from
being engulfed by phagocytes. The efficiency of phagocytosis and killing depends
on the presence of IgG2 anti-capsule antibodies.
Antibodies of the IgG3 subclass are strong activators of the complement system
and bind to all activating Fc-gamma receptors. This makes them strongly pro-
inflammatory. The production of specific IgG3 is generally in parallel with that of
specific IgG1. IgG4 molecules have an unusual feature—they can exchange their
Fab arms in vivo. As a result, they might become bi-specific and their two antigen-
binding sites can bind different epitopes. Such antibodies lose the ability to cross-
link antigens and the avidity of their antigen binding decreases considerably. The
biological role of this phenomenon remains poorly understood. IgG4 antibodies are
often produced in parasitic diseases as well as after the repeated administration of
the same antigen. This is the case during immunotherapy of allergy by the continu-
ous injection of increasing doses of an allergen. These specific IgG4 antibodies are
often referred to as “blocking” as they compete with IgE with the same specificity
for binding to the allergen, and as a result the immediate hypersensitivity reactions
to it are suppressed or even prevented.
Pharmacopoeia rules oblige immunoglobulin producers to ensure that the ratio
of IgG subclasses in the pooled IgG preparations corresponds to that in healthy
plasma donors. The fractionation technologies used allow this requirement to be
easily met.
11.2.5 IgG Monomers, Dimers, and Aggregates

in Therapeutic Immunoglobulins
Some IgG molecules tend to aggregate in concentrated solutions. While IgG mono-
mers and dimers are normal components of therapeutic immunoglobulin
concentrates for intravenous administration, the percentage of aggregates is strictly

controlled. The “art” of plasma fractionation is to apply technologies that result in
as little aggregation as possible in the final product and which ensure the prevention
of further aggregation during the shelf-life of a preparation.
The immunoglobulin plasma fraction obtained by cold ethanol fractionation in
the early 1940s of the last century has been infused to volunteers. All developed a
severe anaphylactic type of reaction (see Chap. 12). It took 20 more years to define
the responsible mechanism. The drop of blood pressure has been shown to be linked
to the presence of complement-activating IgG aggregates in the intravenously
injected preparation. The activation of the complement system results in the genera-
tion of anaphylatoxins (C3a and C5a, released from the parent molecules). Both
anaphylatoxins trigger the release of pre-formed mediators from basophils and mast
cells. The mediators released (mainly histamine) cause dilatation of postcapillary
venules, increased permeability of blood vessels, and hypotension.
IgG dimers are a regular component of all therapeutic immunoglobulins. They
form spontaneously when two (or more) IgG molecules engage in, e.g., idiotype/anti-
idiotype binding (Roux and Tankersley 1990). The affinity of these interactions is low
and dimers take time to form. The percentage of dimers increases with the number of
individual donors the plasma pool for fractionation comes from, as well as after stor-
age of the IgG solution at 4 °C. The optimal pH for their formation is around 7.
The intravenous infusion of a preparation with a dimeric fraction above 8–10%
has been shown to cause a cytokine storm in healthy volunteers (Späth et al. 2015).
Control of dimer formation was achieved through L-proline at moderately lowered
pH of the liquid IVIg (Bolli et al. 2010).
11.2.6 Non-immunoglobulin Molecules

in Therapeutic IgG Preparations
Immunoglobulin preparations from different manufacturers may contain sugars

(sucrose, maltose, etc.) or amino acids (glycine, L-proline, L-isoleucine, etc.),
added to improve IgG long-term stability and diminish the formation of IgG aggre-
gates and dimers.
In addition to pooled IgG, therapeutic immunoglobulins contain some amounts of
human serum albumin, IgM and IgA, traces of IgE as well as fragments from immu-
noglobulins, and other plasma proteins. Their contribution to the overall antibody
repertoire of the preparation is negligible. Anti-IgA antibodies are found in about
25% of individuals with selected IgA or IgA plus IgG deficiency. Non-IgE-mediated
anaphylactic reactions may develop in them after IVIg infusions. R. Rachid et al.
report that out of 22 IgA-deficient patients, three had anti-IgA antibodies and only
one of them has had an anaphylactic reaction in the past. All of the studied patients
tolerated subcutaneous administration of immunoglobulin (Rachid et al. 2011).
Numerous research groups have used sensitive analytical methods to look for the
presence of non-immunoglobulin human molecules in therapeutic IVIg—cytokines,
HLA and CD molecules, etc. (Blasczyk et al. 1993; Lam et al. 1993; Sherer et al.
2001). The existence of traces of these molecules comes as no surprise as most of

them are members of the big immunoglobulin superfamily. Authors of these publi-
cations generally speculate that the non-immunoglobulin molecules may contribute
to some of the immunomodulating effects of the therapeutic immunoglobulins. The
concentration of these impurities is, however, low, and they can hardly be expected
to have a biological effect.
11.2.7 Specific High-Titer Immunoglobulin Preparations
While most immunoglobulin preparations used today contain pooled human IgG
isolated from plasma of healthy donors, there is still a small niche for specific high-
titer immunoglobulins of animal (mostly equine) origin, as well as increasing niches
for high-titer preparations of polyclonal human IgG as well as of human monoclo-
nal immunoglobulins for passive prophylactic immunization and immunotherapy of
infectious diseases.
Normal pooled IgG preparations contain antibodies neutralizing various bacte-
rial toxins, viruses, etc. The levels of the respective protective antibodies in indi-
vidual production lots are only known when being parameters of lot release.
High-titer preparations are from reconvalescent plasma, from plasma of immunized
volunteers, or from pre-screened individual donors. The levels of respective anti-
bodies are standardized and the optimal doses to be administered are well known.
A hundred and twenty five years after the first use of hyperimmune horse serum
in children with diphtheria, animal immunoglobulins are still used for the treatment
of patients with diphtheria, tetanus, food-borne botulism, venomous snakebites, etc.
The side effects and dangers of injecting animal antibodies to humans are serious and
very well known. Their half-lives are short (approx. 5 days) and the lifelong sensiti-
zation to the administered animal proteins is inevitable. These preparations have an
additional disadvantage—they could not be injected intravenously, but only intra-
muscularly. Passive immunization is an emergency procedure which works well
when the antibodies are injected intravenously and reach the tissues quickly (Bayry
et al. 2004). The reason for the continuous production and use of animal immuno-
globulins is that they are cheaper and more easy to produce, while their human ana-
logs are expensive, are not available worldwide, or are even nonexistent.
Several specific high-titer human immunoglobulins are available for intravenous
administration: anti-D, anti-tetanus, anti-cytomegalovirus, anti-botulism, etc.
(Lazarus et al. 1998); Arnon et al. 2006; Alexander et al. 2010). A human specific
intravenous immunoglobulin for the treatment of Crimean/Congo hemorrhagic
fever has been shown to be effective in a small clinical trial (Vassilenko et al. 1990),
but its orphan drug status prevented its further production. The therapeutic potential
of reconvalescent serum and monoclonal anti-Ebola virus monoclonal antibodies
for Ebola virus disease has been proved in recent epidemic in sub-Saharan Africa
(Moekotte et al. 2016).
A good recent example for the successful use of a human monoclonal antibody in
a bacterial-caused disease is that of bezlotoxumab in severe Clostridium difficile
infection. The administration of this antibody, specific for the A toxin of the pathogen,
has resulted in a significantly lower rate of recurrent infections (Wilcox et al. 2017).
11.3 Antibody Repertoires in Therapeutic Immunoglobulins
11.3.1 Anti-protein Antibodies in Therapeutic Immunoglobulins
The extremely diverse repertoire of antibody specificities in each production batch

of immunoglobulins keeps track of all previous immune activities during the life-
time of the plasma donors (Lacroix-Desmazes et al. 1995; Fesel and Coutinho
1999). The techniques for the indirect antibody repertoire profiling have evolved
from immunoblot through phage libraries to microarrays.
In healthy people the total IgG reactivity with bacterial antigens was shown to be
highly diversified both between individuals and as a function of age (Lacroix-
Desmazes et al. 1995). Furthermore the repertoire can be probed through immuno-
globulin variable region gene sequencing. It can also be explored by the combined
profiles of binding to known antigens or to the proteome. Studying therapeutic
immunoglobulin antibody repertoires in detail can detect unexpected antigen-
binding specificities which might have therapeutic relevance. The patterns of bind-
ing to large arrays of mimotopes observed in the sera of patients have been referred
to as immunosignatures. They have been shown to carry information beyond that
provided by defined sets of well-known antigens (Merbl et al. 2009).
Thus, probing the entire Ig repertoire is more than just exploring a bunch of
humoral immune responses. Natural antibodies (of IgG, IgM, and IgA isotypes) bind
a variety of xeno- and autoantigens in a polyspecific manner (Fig. 11.2). Even with-
out an antigenic stimulation, they are an essential, nonredundant factor of the defense
against viral infections (Baumgarth et al. 2000). On the other hand, induced or adap-
tive anti-protein antibodies are not only elicited by specific antigens, but they are also
T cell-dependent and, thus, of high affinity and high specificity. In most repertoire
binding assays, it is hard to distinguish natural from induced antibodies.
11.3.2 The Repertoire of Carbohydrate-Specific

Antibodies in IVIg
The surface of all living cells is covered by a layer of complex carbohydrates (gly-
cans) also referred to as glycocalyx. Exposed to the cellular environment, these car-
bohydrates, often attached to glycoproteins or glycolipids, harbor multiple functions
including charge repulsion effects, ligand-receptor interactions, adhesion, immuno-
logical identification (e.g., blood group antigens), and protection. Microbes often
carry characteristic glycan structures, such as contained in bacterial cell walls or
capsules, which are exploited for diagnostic purposes or as targets of antimicrobial
therapy. In multicellular organisms there are species-dependent and interindividual
differences of glycosylation, with carbohydrate xenoantigens and blood group
DNAJA4
KIF19
LAX1
PLEKHM2
H2AFY
PRKAR2B
ODC1
IGHG1
CFAP36
EPCAM
NoI3
Nc2b
Lin28a
THOP1
MASP1
SH3BGR
VWA8
IBSP
Fig. 11.2 Typical IVIg reactivity with a human protein array. Multiple natural anti-self reac-
tivities are observable. Human proteome arrays (HuProt™, Cambridge Protein Arrays) were
treated with 0.7 μM IgG from native or Fe2+-exposed IVIg. Only block #19 (out of 24 blocks)
is shown as an example. The labeled proteins indicate that the natural reactivities include not
only sequestered intracellular proteins but also some receptors and secreted proteins (see
Table 11.2)
determinants providing challenges to the fields of transfusion and transplantation

medicine. Furthermore, intraindividual differences in cell surface glycosylation pat-
terns exist, which explains the organotropism of certain microbes. Aberrant surface
glycosylation is observed in essentially all human malignancies, and so-called tumor-
associated carbohydrate antigens (TACA) have not only diagnostic significance but
Table 11.2 Identity and function of the self-proteins bound by IVIg (see Fig. 11.2)
Gene Protein name Notes
DNAJA4 DnaJ heat shock protein Heat shock protein
family (Hsp40) member
A4
LAX1 Lymphocyte Membrane, regulator or TCR and BCR
transmembrane adaptor 1 signaling
KIF19 Kinesin family member 19 Role in platelet development, motor
protein in cilia
PLEKHM2_frag Pleckstrin homology and Golgi, membrane movements,
RUN domain containing salmonella-induced filaments
M2
H2AFY H2A histone family Medulloblastoma antigen
member Y MU-MB-50.205
PRKAR2B Protein kinase CAMP-
dependent type II
regulatory subunit beta
ODC1 Ornithine decarboxylase 1
CFAP36 Cilia- and flagella-
associated protein 36
EPCAM Epithelial cell adhesion Tumor-associated GI tract, interaction
molecule, CD326 between intestinal epithelial cells and
intraepithelial lymphocytes
Nol3 Nucleolar protein 3 Antiapoptotic
Nc2b Downregulator of Chromatin organization
transcription 1
Lin28a Zinc finger CCHC Posttranscriptional regulator of genes
domain-containing protein involved in developmental timing and
1 self-renewal in embryonic stem cells,
tumor antigen
THOP1 Thimet oligopeptidase 1 Cleaves cytosolic peptides, making them
unavailable for display on antigen-
presenting cells
SH3BGR SH3 domain-binding Skeletal muscles, myocytes, proline rich,
glutamate-rich protein glutamate rich
MASP1 Mannan-binding lectin Lectin pathway of C′
serine peptidase 1
VWA8 von Willebrand factor A
domain containing 8
IBSP Integrin-binding Rich in bone. Acidic amino acid clusters
sialoprotein
also influence tumor progression (Boligan et al. 2015). IVIg contains reactivity to an
extensive range of complex carbohydrates (Schneider et al. 2015). Recognized gly-
cans included certain blood group antigens, tumor and microbial antigens, including
bacterial cell wall, and capsular or exopolysaccharides. IVIg reactivity is not
restricted to microbes, but include bacterial and viral glycan receptors (attachment
sites), as well as carbohydrate tissue attachment sites of the exotoxins Shiga toxin
from Shigella dysenteriae type 1 and the verotoxins SLT-I and SLT-II from
Blood group antigens Tumor antigens
Xenoantigens Lectin ligands
Microbial structural Anti -carbohydrate Bacterial and viral

antigens antibodies in IVIG attachment sites
Microbial Attachment sites

exopolisaccharides of exotoxins
Other glycans
Fig. 11.3 Overview on proposed carbohydrate antigens (rectangles) recognized by glycan-

specific antibodies in intravenous immunoglobulin (IVIg)
Escherichia coli, suggesting a potential role of glycan-specific antibodies by prevent-

ing adhesion processes and sequelae of infection (Schneider et al. 2015).
The comparison of different intravenous and subcutaneous immunoglobulin prep-
arations using glycan array technology revealed a universal architecture of the human
IgG anti-carbohydrate repertoire. A striking association between IVIg- binding
capacity and terminal carbohydrate moieties was found, showing an association
between structural features and glycan immunogenicity or tolerance (Schneider et al.
2015). Textbooks often refer to glycans as only poorly immunogenic and T cell-
independent antigens. However, studies of the anti-carbohydrate repertoire indicate
that class-switched IgG antibodies in IVIg, including non-IgG1 IgG antibodies, rec-
ognize a broad variety of glycans, thus indicating T cell involvement (Fig. 11.3). In
line with IVIg-derived data, in a recent system biology study, the meningococcal
polysaccharide vaccine MPSV4 was shown to induce higher IgG titers than a conju-
gate vaccine, and the analysis of blood transcription modules (BTM) revealed evi-
dence for T cell signaling (Li et al. 2014). Furthermore, there is evidence for
carbohydrate-specific T cells, also referred to as “Tcarbs,” that specifically recognize
presented carbohydrate epitopes from glycoconjugate vaccines (Avci et al. 2011).
Research on the repertoire of carbohydrate-specific antibodies in IVIg revealed more
insights into the composition and mechanisms of IgG concentrates as pluripotent
drugs (Pashova et al. 2017).
11.3.3 Anti-idiotype Antibodies
Immunoglobulins are big, variable, and complex molecules and—therefore—

immunogenic. Their antigenic determinants (epitopes) are divided into three
groups—isotypic, allotypic, and idiotypic. The isotypic ones are present in all mem-
bers of a given immunoglobulin class or subclass. The allotypic ones are inherited
as alleles and each person inherits particular allotypes.
The idiotypic determinants (idiotopes) are unique antigen determinants of an
antibody variable region. The idiotype of an individual antibody molecule is the
combination of all its idiotopes. All antigen-binding molecules, free circulating
immunoglobulins, membrane-bound immunoglobulin receptors of B cells, as well
as T cell antigen receptors, have idiotopes. The latter can be “public”—when they
are shared between several antibody clones— or “private” when they are unique for
the antigen receptor of a cell and the antibodies produced by its progeny.
Immunoglobulin preparations contain a vast diversity of IgG molecules that can
network by idiotype-anti-idiotype interactions. This results in the formation of
dimers during the shelf-life of a liquid preparation (see above).
Niels Jerne (Nobel prize for 1984) has hypothesized in 1974 that the self-
recognition of antigen-binding cell receptors and circulating immunoglobulins based
on idiotype/anti-idiotype interactions ensures their connectivity and is the main con-
trol mechanism in the immune system. According to this “network” theory, the
immune system is self-controlled by using signals generated within itself. The
immune response to an antigen is considered as a temporary imbalance between the
antigen-specific clone and the anti-idiotypic clones (Jerne 1974). Autoimmune dis-
eases are believed to be the result of defective self-control of the immune network.
The network hypothesis might in part explain the beneficial effects of infusions of
large doses of normal pooled IgG to patients with autoimmunity. Beneficial effects
are provided by the introduction of components of a healthy immune network that
helps repair their dysfunctional ones. The philosophical beauty of these ideas is evi-
dent. However, quarter of century ago, the interest in them faded after the discovery
of the main mechanisms of immune tolerance and memory. The new knowledge
rendered the immune function explanation by network emergent properties largely
dispensable.
Nevertheless, discarding idiotype interactions as biologically irrelevant may be
premature. Their role in the beneficial effects of pooled immunoglobulins cannot be
ruled out. A single IVIg infusion to patients with autoimmune hemophilia has
resulted in the rapid disappearance of 95% of the disease-associated anti-factor VIII
antibodies. The effect was proven to be due to the binding of anti-idiotype IgG anti-
bodies from the administered preparation to the idiotypes of pathological IgG with
anti-factor VIII reactivity in the patients (Rossi et al. 1989).
11.3.4 Antibodies with Natural (Innate) and

with Induced Polyspecificity
The father of immunochemistry Karl Landsteiner has proven 90 years ago that anti-
bodies can bind an antigen with exclusive specificity, but he has never claimed that all
antibodies are monospecific. In the next five decades, however, that was the conven-
tional paradigm. The ability of an individual immunoglobulin molecule to bind to two
or more structurally unrelated antigens was formally proven only by studying
monoclonal immunoglobulins. Monoclonal antibodies were produced using the prog-

eny of a single malignant B cell or clones of engineered single B cells, constructed
using the hybridoma technology. A large percentage of the B cell clones produced IgG
antibodies able to bind more than one antigen. While all IgM were polyspecific, only
part of those belonging to the IgG, IgA, and IgE isotypes displayed such antigen-
binding promiscuity. Interestingly, there is still no universally accepted definition of a
polyspecific antibody. The proof of binding to two or more foreign or self-antigens
that are structurally unrelated is regarded so far as sufficient to classify an antibody as
being polyspecific. Yet, may be any antibody could be shown to interact with two or
more antigens were the testing panels large enough (Van Regenmortel 2014).
Polyspecific antibodies are often neglected as “background,” “silent,” “sticky,” etc.
Recent studies have proven, however, their important role in the first line of innate
defense against the dissemination of pathogens in the pre-immune host. The knowl-
edge on the biophysical mechanisms of polyspecific antigen binding by IgG antibodies
and their biological properties have been recently summarized (Dimitrov et al. 2013).
Apart from “innate,” the polyspecificity of IgG molecules can be also
“induced.” Such antibodies are referred to as “masked,” “hidden,” “cryptic” or
“latent.” The IgG exposure to acid pH buffers is known to result in enhanced
polyspecificity (Bouvet et al. 2001). This phenomenon may not be directly
related to in vivo events. However, some of the licensed therapeutic intravenous
immunoglobulin preparations are produced utilizing a protein fractionation stage
at acid pH used as a supplemental virus-neutralizing and IgG aggregation-pre-
venting step. They have enhanced antigen-binding polyspecificity (see Fig. 11.4)
1 2 3 4 5 6 7 8
Fig. 11.4 Immunoblot analysis of the reactivity of seven different licensed IVIg preparations to
human liver antigens (the first two are produced using a fraction stage at acid pH). 1 human serum,
2 Octagam, 3 Sandoglobulin, 4 Gammagard S/D, 5 Endobulin S/D, 6 Intraglobin F, 7 Immunovenin
Intact, 8 Venimmun N (from Scand. J. Immunol. (2005), 61, 357)
that includes the newly acquired binding to at least one pro-inflammatory cyto-
kine—interferon gamma. The comparative study of the effects of passive immu-
notherapy in mice with bacterial lipopolysaccharide- induced systemic
inflammation (endotoxemia) has shown that while the administration of intact
IVIg had no effect on survival, a single dose of the same preparation, exposed to
a pH 4.0 buffer, significantly decreased the mortality (Djoumerska et al. 2005;
Djoumerska-Alexieva et al. 2010). This is an important observation, as it points
to the fact that commercially available immunoglobulin preparations may have
different antigen binding as well as different therapeutic properties. To the best
of our knowledge, no clinical trials have been conducted so far to compare the
therapeutic effects of “unmodified” IVIg and preparations exposed to acidic con-
ditions during its production.
Reactive oxygen species (ROS), ferrous ions, and free heme are aggressive
protein-modifying molecules that are released in vivo at sites of inflammation,
severe trauma, hemolysis, etc. This led us to formulate the hypothesis that the circu-
lation of IgG molecules through a local inflammation site might modify their
antigen-binding behavior. The experimental testing fully confirmed this predic-
tion—both in vitro (Dimitrov et al. 2006, 2007) and in vivo (Mihaylova et al. 2008).
Sepsis and other aseptic severe inflammatory response syndromes (SIRS) are
medical disasters that respond poorly to available treatments. The unfavorable
prognosis of these patients is now believed to be due to the quick and dramatic
change in gene expression affecting more than 80% of all cellular functions and
pathways referred to as “genomic storm” (Xiao et al. 2011). This “storm” could
well explain the failure of treatment approaches targeting single inflammation-
related molecules. Passive immunotherapy with pooled IgG is a logical therapeutic
approach because of its broad specificity that encompasses many pathogens—as
well as self-antigens. The results from clinical trials on patients with SIRS are,
however, inconclusive. If the anti-inflammatory effects of IVIg are partially attrib-
utable to its polyspecific natural antibody reactivity, could it be that by additionally
enhancing the polyspecificity would improve its effectiveness? IVIg, preexposed
in vitro to pro-oxidative ferrous ions, was used for the passive immunotherapy of
mice with experimental sepsis or aseptic SIRS induced by the injection of bacterial
lipopolysaccharide (LPS), of live E. coli, of zymosan or by the colon puncture and
ligation technique. The low single dose of only 50 mg/kg of the modified prepara-
tion but not of the native, commercially available IVIg significantly increased sur-
vival in all models of severe generalized inflammation (Fig. 11.5). IVIg exposed to
pro-oxidative ferrous ions was more efficient than the acid pH-exposed IVIg
(Djoumerska-Alexieva et al. 2015).
The intravenous injection of pooled human IgG antibodies to the treated patient
ensures the administration of the quasi-complete IgG antibody repertoire of a big
healthy donor population. There are many questions regarding this treatment that
are still waiting to be answered and mysteries to be solved. One is sure—the better
understanding of the mechanisms of action of normal human immunoglobulins will
result in the optimization of presently used therapeutic approaches and in increasing
the range of potential new ones.
100 100 100
75 * 75 75
% Survival
* *
50 50 50
25 25 25
0 0 0
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
Days after sepsis induction
Fig. 11.5 The administration of a single dose of IVIg with additionally enhanced polyspecificity
improves survival in experimental sepsis and aseptic severe inflammatory syndromes. Survival
curves in endotoxemia (left panel), zymosan-induced systemic inflammation (middle panel), and
polymicrobial sepsis (right panel). Animals were injected intravenously with a single dose of the
native IVIg (black triangles), of the Fe(II)-exposed IVIg (black circles), or with PBS pH 7.4 (open
squares), *p < 0.05, Mantel-Haenszel log-rank test (from Molec. Med. (2014), 21, 1002)
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Essentials of the Production of Safe
and Efficacious State-of-the-Art 12
Polyclonal IgG Concentrates
Peter J. Späth
12.1 Introduction
From the very beginning of the clinical use of plasma-derived protin concentrates
severe noninfectious adverse events (AEs) and transmission of pathogens by plasma-
derived protein concentrates were threats to recipients (see Chap. 10 for additional
information). First anti-infectious plasma potein concentrates were the “standard
IgG” preparations. They were produced by the cold-ethanol fractionation methods
and did not make an exception to the above: noninfectious severe AEs occurred
while infectious AEs were rarely reported. Indeed, prior to the introduction of mass
screening for infection markers of plasma donations, inadvertent transmission of
HIV to recipients of factor VIII and factor IX concentrates did occur, while IgG con-
centrates obtained from the same plasma pool did rarely transmit HIV (Morgenthaler
2001). Rare transmissions were restricted to products not exposed to low pH. The
very few incidences of HIV and some incidences of HCV transmission by IgG con-
centrates in the early 1990s, together with many cases of coagulation factor concen-
trates transmitted viral disease, clearly demonstrated the need to establish standardized
measures to render plasma products pathogen safe. In the second half of the 1990s,
authorities shifted regulatory emphasis from a scientific review of the processes to a
focus on compliance to current good manufacturing practice (cGMP). The focus on
cGMP compliance was applied to all aspects of plasma fractionation and the clinical
use of plasma products. Court injunctions and warning letters were the consequences
of this paradigm shift by authorities. This in turn resulted in a paradigm shift how the
modern plasma industry operates (Steinhardt 1998).
The strict implementation of the recommendations by authorities resulted in
today’s immunoglobulin concentrates in general being well tolerated and safe
regarding the transmission of known blood-borne viruses, the agent of the
P.J. Späth

152 P.J. Späth
Main sequences Measures to achieve

Level Side fractions of fractionation pathogen safety
A Pooled and tested Plasma Donor questionnaire

Mass screening
Cryoprecipitate
Cryo-poor Plasma Testing of pooled plasma
B
Albumin Lipid removal
C1-INH, others Validation of (existing)
C Cold ethanol Ion-exchange fractionation steps
chromatography
Introduction of
Fractions containing dedicated steps
IgA and IgM, others Crude IgG concentrate
Validation of (existing)
D Anti-A and -B Polishing steps to polishing steps
(for chromatographically achieve i.v. tolerability
manufactured IgG) or other particular properties Introduction of
dedicated steps
E Final formulation and shipping
Recovery and Purity
Fig. 12.1 Strongly simplified outline of fractionation of pooled plasma to pathogen free, well-
tolerated IgG concentrates. Side fractions of the process are starting material for plasma products
other than IgG. Level A of manufacturing: generating a large volume of plasma with optimal
safety. Level B: plasma deprived of cryoprecipitate that contains relevant blood-clotting factors.
Level C: a series of steps resulting in a crude IgG concentrate that is not tolerated intravenously.
Level D: a sequence of steps rendering an IgG concentrate clinically well tolerated. Level E:
lyophilized products are adjusted to the appropriate concentration, the excipient is added, the solu-
tion is filled into the vials, and the product is lyophilized. Liquid formulations are finalized simi-
larly while leaving the freeze-drying process. Bottles with lyophilizate or IgG solutions are labeled
and are ready for shipping
transmissible spongiform encephalitis (TSE) or even emerging and reemerging zoo-

notic viruses. Furthermore, moden IgG concentrates have an excellent noninfec-
tious adverse event record, particularly when used in chronic conditions.
Manufacturing of IgG concentrates is a flow of processes as outlined in Fig. 12.1.
Along this flowchart, particular measures can serve to obtain clinical tolerability
and to achieve reduction in pathogens potentially contaminating the starting mate-
rial. Below the efforts undertaken to provide for patients IgG concentrates safe in all
aspects are outlined.
12.2 Plasma Fractionation: Regulatory Agencies’ Requests
Plasma protein concentrates, typically prepared from plasma of thousands of

donors, inevitably are associated with the risk of transmitting blood-borne patho-
gens (see Chap. 10). Therefore, modern fractionation of human plasma for clinical
use is embedded in a tight network of quality assurance (QA) put in place by recom-
mendation of authorities. Good manufacturing practice (GMP) is part of a QA pro-
gram (Slopecki et al. 2007). GMP is a guidance for industry. GMP ensures
12 Essentials of the Production of State-of-the-Art Polyclonal IgG Concentrates 153
consistency in production. It describes the minimal requirement a product must

fulfill for obtaining a marketing authorization and covers procedures for receipt of
materials, production, packaging, labeling, quality control, release, storage, and dis-
tribution. The procedures and laws of GMP are defined by each country. GMP is a
“moving target” and current GMP (cGMP) includes the most recent developments
and knowledge of the field; as technology and information capabilities change, so
do the requirements for maintaining good manufacturing practices. On a solid fun-
dament of GMP, the elements for a state-of-the-art plasm product are:
• Good clinical practice (GCP).

• Good laboratory practice (GLP).
• Standard operation procedures (SOPs).
• Training of staff.
• Donor selection, deferral and inventory hold.
• Mass screening of donation for infection markers by serology and nucleic acid
amplification technology (NAT) testing. Recently one company added isoagglu-
tinin titer screening in plasma donations in order to obtain plasma pools for chro-
matographic fractionation low in isoagglutinins anti-A and anti-B (Siani et al.
2014).
• In-process controls (production parameters and infection markers).
• Validation studies (manufacturing steps and dedicated pathogen reduction).
• Polishing steps (clinical tolerability and pathogen reduction).
• Labeling and shipment.
• Cleaning and segregation.
• Look back.
• Pharmacovigilance.
• Audits by internal and external inspectors.
A plasma fractionation, which is compliant with GMP, has undergone process

validation for each manufacturing step. Process validation helps to ensure that sys-
tems are performing in the intended manner. Process validation ensures that the
product has the required potency, purity, and safety. The parameters of a single
process step are defined on a laboratory scale. The parameters obtained during the
validation process help to define the elements of process control and provide a
mechanism for ongoing quality assurance, quality control, training, competency
review, and continuous improvement (Preti 1999).
Ensuring pathogen safety of a plasma product needs a tripod of measures: (1)
donor selection, (2) mass screening for pathogen markers, and (3) pathogen elimi-
nation and inactivation during manufacturing. As an example how to implement
pathogen safety of plasma products, the view of the European Medicines Agency is
cited: “The safety of a product has to be demonstrated experimentally by validation
studies on a laboratory scale. Such studies are based on the principle of deliberate
contamination of the intermediate product by model viruses at given stages of the
manufacturing process and monitoring the reduction in infectivity produced by the
154 P.J. Späth
production step under consideration.” At the time when GMP was introduced, well-
established fractionation procedures were in place. For validation studies, the pro-
cesses had to be downscaled to a laboratory scale. In a first step, it had to be
demonstrated that the scaled-down process mimics the process in production, i.e.,
ideally has identical process parameters. Furthermore, authorities request at least
two “dedicated” virus removal/inactivation steps in sequence during the fraction-
ation. The two methods should represent two different principles of action thereby
assuring that virus reduction is complementary.
When fractionating plasma on the fundaments of cGMP and applying the “full
package” of complementary recommendations a state-of-the-art immunoglobulin
preparation results.
Any activity from plasma collection to delivery at the door of a customer that is
not performed and documented according to the standards may have severe conse-
quences to the noncompliant company. Deficiencies in adherence to GMP detected
during inspections of companies can lead to consent decrees, and severe deviations
can have the consequence of a company forced to cease distribution of its products
(http://www.ema.europa.eu/docs/en_GB/document_library/Press_release/2010/09/
WC500097037.pdf; https://wayback.archive-it.org/7993/20161023082812/http://
www.fda.gov/mw/ucm223968.htm; both accessed April 2017).
The leading regulatory agencies, which are enforcing the adherence to QA frac-
tionation recommendations, are the US Food and Drug Administration (FDA;
https://www.fda.gov/) and the European Medicines Agency (EMA; www.ema.
europa.eu), while in all countries own agencies govern the enforcement of recom-
mendations. A list of regional offices for Africa, for the Americas, for the Eastern
Mediterranean, for Europe, for Southeast Asia, and for the Western Pacific can be
found under: http://www.who.int/medicines/areas/quality_safety/regulation_legis-
lation/ListMRAWebsites.pdf (accessed April 2017).
12.3 P
lasma Collection: The Starting Point for Quality
and Safety of a Plasma-Derived Product
IgG concentrates belong to the stable blood products. Ensuring quality of an IgG
concentrate starts with collection of plasma, its correct handling, and cautious
pooling.
Donor selection (level A, Fig. 12.1): Plasma fractionated to current best prac-
tice IgG concentrates all are from donors donating blood or plasma from free will.
There are two types of donations: whole blood from which plasma is collected
after centrifugation (recovered plasma) or plasma obtained by apheresis device
(apheresis or source plasma) (Table 12.1). There is epic, for several decades’
ongoing debate which of the two ways of plasma collection is of higher ethical
and better biological quality. There is no reason to step into this discussion in this
book chapter. However, it has to be acknowledged that apheresis plasma collec-
tion of volumes at the upper end of what is allowed might have an influence on
protein composition of the donated plasma (Laub et al. 2010). As GMP covers
minimal requirements for quality, and because having been accused remunerated
Table 12.1 Some characteristics of collection of plasma for fractionationa

Recovered plasma Apheresis plasma
Collection method Whole blood donations; nothing is Plasmapheresis; the cellular
returned to the donor’s circulation; components are returned to the
plasma is separated by donor by continuous machine-
centrifugation and the cellular aided separation of plasma
components serve therapies by form blood cells
these “labile blood products”
Remuneration Predominantly none (especially in Most (especially in the USA)
the European Union)
Collection frequency Max. every 2 months Intervals differ in various
and max. number of regions of the world
donations per year 4–6 USA: up to 104b
EU: 15–50b
Collected volumes of Approx. 250 mL Approx. 600 mL
plasma in one session 1.5 L (including anticoagulants) USA: 90 Lc
and allowed maximal EU: 10–35 Lc
volume per year
Extent to which 20–25% 75–80%
worldwide plasma
requirements are
covered
a
Donor suitability is defined by national regulatory authority requirements for whole blood dona-
tion plus additional special requirements for plasma donation
b
Donation is allowed when total plasma protein content is >60 g/L (the USA, Germany) and when
level is IgG > 6 g/L (Germany)
c
EU: to be assessed after every fifth donation; USA, serum protein electrophoresis every 4 months;
the protein quality of donations from donors donating very high volumes per year having low
plasma protein content has been addressed recently (Laub et al. 2010)
donations being of lesser quality and safety, the apheresis plasma collecting and
fractionating industry has introduced additional voluntary regulations. These are
the International Quality Plasma Program (IQPP) introducing qualified donor
standard, implementation of a donor deferral registry, and a drug abuse screening
and the Quality Standards of Excellence, Assurance, and Leadership (QSEAL).
QSEAL covers control on incoming plasma, inventory hold, NAT testing, inter-
mediates purchased form external suppliers, recovered plasma specification, qual-
ified donor, and viral markers (http://www.pptaglobal.org/safety-quality/
standards/qseal; accessed June 2017). With the implementation of these voluntary
standards, no relevant differences are found in pathogen marker frequencies of
either type of plasma donation (EMEA 2002). In summary, the main goal of donor
selection is to ensure that donors with high risk are excluded form donating, or
their donation is withheld from further processing (Table 12.2). The donor is
informed if HIV positive.
Finally yet importantly, plasma donations require careful handling to prevent
formation and accumulation of vasoactive (e.g., prekallikrein activator (PKA)) or
coagulation promoting (e.g., coagulation factor FXIa) substances. Due to their
physicochemical properties, their removal during the manufacturing process may
remain imperfect, and they may induce severe adverse events.
156 P.J. Späth
Table 12.2 Outline of the main measures of safe donor selection

• Physical examination
• Donor questionnaire, confidential self-exclusion
Detailed health history questionnaire relative to current known safety risks, designed to
obtain information, e.g., about acute infections, (silently progressing), chronic illness,
or elevated risk, for having acquired blood-borne pathogens (e.g., social behavior,
sexually transmitted diseases, or having had tattoos, acupuncture, or ear/body piercing
in the past 12 months) or having received xenotransplants (cornea) of animal origin or
having been treated by pituitary (growth) hormones of human origin
• Donor deferral, e.g., donors having traveled or lived in endemic regions; donor deferral is a
moving target (Yang et al. 2017)
• Inventory hold, e.g., allow for the retrieval of plasma prior to use if new post-donation
information becomes available regarding the donor’s health status
The apheresis plasma collecting industry has in addition established the IQPPa and QSEALb
standards that in addition to the above add
1. Qualified donor standard; donor successfully passing two donor medical history
screenings and required viral testing
2. Community-based donors; only donors accepted with proof of a permanent address
3. The use of PPTA Source’s National Donor Deferral Registry (NDDR) helps ensure that
deferred donors cannot donate their plasma again, e.g., at another center
4. Education of donors at risk for infectious disease; education how to avoid behavior that
is believed to lead to an increased risk of (HIV) infection
5. Personnel education and training standards
6. Professional medical facility criteria
7. Quality assurance
8. Viral marker standard
As 2/3 of plasma for fractionation is obtained from remunerated plasmapheresis donations, addi-
tional voluntary standards have been introduced by the corresponding plasmapheresis collecting
industry to minimize the risk of infections remunerated donations might harbor
Donor deferral can be established, e.g., a donor has lived for a certain time in a given region of the
world and has received a xenotransplant, a drug (D’Aignaux et al. 1999) or medical devices with
parts of animal origin. Donor deferral is a moving object (Yang et al. 2017)
a
IQPP International Quality Plasma Program
b
QSAEL Quality Standards of Excellence, Assurance, and Leadership; a certification program to
which donation centers have to comply
Mass screening of donations (level A, Fig. 12.1): Mass screening is the measure

to exclude donations potentially contaminated by pathogens such as virus(es).
Stable blood products inherit a particular risk for transmission of viruses because
the staring material being a pool of thousands of individual donations (Table 12.3).
Parameters of mass screening for virus markers are outlined in Table 12.4 and
include serological and nucleic acid amplification technology (NAT) testing. NAT
testing in addition to serological screening was introduced in order to shorten the
time period of “window donations.” During a window period, a donor already har-
bors the infective agent and is infective while seroconversion has not occurred yet,
i.e., antibody formation has not set in, or antibodies are at a titer not detectable by
validated serological methods. By introducing NAT testing, the estimated risk of an
undetected infectious donation entering the blood supply dropped as given in
Table 12.3 Some human pathogens transmissible or theoretically transmissible by blood and
plasma productsa
Suspected cases of
transmission most
likely through
non-leukocyte-
Ascertained depleted red blood
Ascertained transmission transmission by cell concentrates or
by blood or plasma blood or plasma contaminated
products with possibly products with no No report of plasma-derived
severe clinical known clinical transmission by factor VIII
consequences consequences transfusion concentrate
Hepatitis B virus Hepatitis D virus TSE agent of classical/ TSE agent of the
(enveloped, HBV) (enveloped) sporadic CJD (sCJD)d,g variant form of
CJD (vCJD)d,f
Hepatitis C virus Hepatitis F virus Severe acute respiratory
(enveloped, HCV) (non-enveloped) syndrome-associated
coronavirus (SARS-
CoV, enveloped)
Human immunodeficiency GBV-C/hepatitis Chikungunya virus
virus 1/2 (HIV 1/2, G virus (CHIKV, enveloped)
enveloped) (enveloped)
Human T cell leukemia SEN virus
virus HTLV I/II (enveloped)
West Nile virus (WNV, TT virus
enveloped) (enveloped)
Hepatitis A virus
(non-enveloped, HAV)
Hepatitis E virus (HEV,
non-enveloped)b
Erythrovirus B19 (B19V,
non-enveloped)c
Zika virus (ZIKV,
enveloped)e
a
After implementation of virus reduction steps in the fractionation process of plasma-derived
medicinal products and thorough the enforcement by authorities of the rigorous implementation of
good manufacturing practice (GMP) rules, no further proven transmission of (enveloped) viruses
was reported. Criteria invariably present in true virus transmission by stable plasma products are
(1) several patients infected with the same virus, (2) cluster of transmission (e.g., a restricted num-
ber of hospitals), (3) it is possible to identify one/a few lots that were used in all of the affected
hospitals, and (4) the same virus is identified in the relevant product lot(s) and in patients treated
with these lots. If these criteria are not fulfilled, transmission by plasma products of pathogen
remains uncertain
b
HEV infection by genotypes 1 and 2 cause large epidemics in in tropical and subtropical regions.
The transmission is via the fecal-oral route. Infections by genotypes 3 and 4 are an emerging threat
in countries of the northern hemisphere and are a food-borne disease. Pigs are the animal reservoir
for this zoonotic virus. These genotypes have been identified in blood donors in Europe. Transfusion
associated with HEV infection was reported (Tamura et al. 2007). Significant acute hepatitis can
be the result of chronic HEV infection in immune compromised patients (Motte et al. 2012), while
in immunocompetent patients, HEV infection has been associated to neurological complications
(continued)
158 P.J. Späth
Table 12.3 (continued)
c
Human parvovirus B19
d
CJD = Creutzfeldt-Jakob disease; aberrantly folded prion proteins (PrPSc) are considered to be the
infectious agent of transmissible spongiform encephalopathy (TSE) diseases, sCJD sporadic CJD,
vCJD variant CJD
e
Reports on transfusion, solid organ transplantation, or sexually transmitted ZIKV disease are
available (Barjas-Castro et al. 2016; Nogueira et al. 2017; Venturi et al. 2016). Prenatal infection
is associated with microcephaly, while infection in adults can be fatal or be associated with
Guillain-Barré syndrome or severe thrombocytopenia
f
The transmission by non-leukocyte depleted erythrocyte concentrates might represent a risk for
transmission. Four possible transmission events by non-leukocyte-depleted erythrocyte concen-
trates have been reported. In one case of probable transmission, the most likely route of infection
was by contaminated plasma-derived factor VIII concentrate
g
In May 2017 a report on two patients receiving blood-clotting factor concentrates dying form
sCJD was posted electronically (Urwin et al. 2017). A causal link between the treatment with
plasma products and the development of sCJD has not been established
Table 12.4 Mass screening on plasma for fractionationa

Pooled plasma
Parameters All individual donations before fractionation
Serological
Anti-human immunodeficiency virus Yes Yes
(HIV)-1/-2 antibodies
Anti-hepatitis C virus (HCV) Yes Yes
antibodies
Hepatitis B virus surface antigen Yes Yes
(HBsAg)
Nucleic acid testing (NAT) by mini-pool
HIV-1 RNA Yes Yes
HCV RNA Yes Yes
HBV DNA Yes Yes
HAV RNA Not mandatory for plasma No
donations for fractionation
B19 Virus DNA Not mandatory for plasma No
donations for fractionation
a
Mass screening for the agent of variant Creutzfeldt-Jakob disease has become feasible; however,
it is not practiced (Jackson et al. 2014)
Fig. 12.2. In addition to the minimal requirements given by GMP, most manufactur-

ers request human parvovirus B19 and HAV NAT testing of plasma donations.
NAT testing is performed in mini-pools and largely can be automated. Aliquots
from bar code labeled pilot tubes are transferred to, e.g., a micro titer plate. In such
an X/Y layout, each aliquot is unequivocally associated to an individual donation.
Starting NAT testing, aliquots from the aliquots are pooled, undergo validated
amplification of part(s) of pathogen’s genome, and the resulting genome equivalents
are quantified. The size of the mini-pool depends on amplification efficiency and
minimal infectious dose of the given pathogen. In case a mini-pool is reactive, pool-
ing is repeated, this time by rows and columns. From the row and the column pool
6.00E+06
5.00E+06
Reciprocal of estimated risk

4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
HIV-1 HBV HCV
Serology With NAT
Fig. 12.2 Estimated risk of an undetected donation entering the blood supply when mass screen-
ing is by serology only or NAT is performed in addition (Offergeld et al. 2005). The reciprocal of
risk rate of undetected infectious donations is given; the higher the bar, the lower the risk rate
being reactive, the X/Y layout allows the identification of the reactive donation. The
contaminated donation is withheld from pooling and is destroyed safely.
Plasma fractionation starts with pooling of individual donations to an appropri-
ate volume. Before starting the fractionation process, an in-process control is per-
formed to ascertain absence of potential contaminating viruses (Table 12.4). This
reduces the risk of pathogen transmission and reduces the financial risk of losing an
entire lot of IgG concentrate.
12.4 F
ractionation Methods in Use to Obtain From Plasma
Well-Tolerated, Highly Pure IgG Concentrates
Over many decades, the development of IgG fractionation was driven to achieve
intravenous tolerability and increase recovery and purity of the concentrates. Today
two different main methods are in place how to fractionate plasma: the cold-ethanol
fractionation and the ion-exchange chromatography process (Fig. 12.1). Before
applying either method, frozen plasma is thawed at 0 °C. The part remaining insol-
uble at 0 °C is separated and represents the cryoprecipitate that harbors the blood
coagulation factors (level A to B in Fig. 12.1). The supernatant is the cryo-poor
plasma that contains a wide array of plasma proteins, including immunoglobulins
and albumin.
Cold-ethanol fractionation is either according to Cohn/Oncley or to Kistler-
Nitschmann (Oncley et al. 1949; Nitschmann et al. 1954) (see Chap. 10 as well).
From cryo-poor plasma, a precipitate is obtained which contains the immunoglobu-
lins (level B, Fig. 12.1). The supernatant is the starting material for albumin frac-
tionation. Further suspension/precipitation steps of the precipitate containing the
160 P.J. Späth
Table 12.5 Polishing steps to achieve particular properties of clinically applicable IgG, e.g.,
intravenous tolerability
• Polyethylene glycol precipitation, removes aggregates
• pH 4 and traces of pepsin; alters aggregates; low pH is a virus inactivating step
• pH 4; virus inactivation; reduction of aggregates
• Depth-/ultra-/diafiltration; not to confound with virus filtration
– Depth filtration uses a filter on which the precipitated protein is separated from the
supernatant using “filter aids.” Filter aids help to maintain high flow rates during the
filtering process and at the same time may adsorb pathogens and or pathogen-protein
complexes quite efficiently: is predominantly applied during cold-ethanol preparation
of crude IgG, and can remove aggregates and adsorb viruses
– Diafitration serves to remove, e.g., salts from an IgG solution with the help of
semipermeable membranes
– Ultrafiltration uses pressure or concentration gradients and semipermeable membranes
• Reduction of isoagglutinins anti-A and anti-B by immunoaffinity chromatography step
(Dhainaut et al. 2013; Höfferer et al. 2015); elevated isoagglutinin titers are associated with
the chromatographic fractionation method; reduction in rate of hemolytic adverse events
immunoglobulins finally end up what is termed “IgG bulk” (level C, Fig. 12.1). This
crude IgG concentrate then undergoes various polishing steps (Table 12.5). Polishing
steps and the final formulation are those differing the most among various compa-
nies (levels D and E, Fig. 12.1).
The early “standard” IgG concentrates underwent only few polishing steps of
the “IgG bulk” material. They contained aggregates that rendered them not tol-
erable when applied intravenously. Typically, these i.m. products were of a solu-
tion strength of 16–16.5%. By introducing virus inactivation/removal steps to
the original manufacturing process, some of these products survived to our
days.
A “first generation” of IgG products for intravenous use (IVIGs) was deprived of
unwanted Fc-effector functions by harsh enzyme treatment. The digested IgG mol-
ecules were heavily impaired in their biological functions however were well toler-
ated. One product still available posted on a Japanese website (see Chap. 13). For a
“second generation” of IVIGs, intact IgG molecules were chemically modified in
order to prevent overt spontaneous complement activation in the recipients’ circula-
tion. Only a very few of these chemically modified products are still available in
some regional markets. They are mentioned in Chap. 13 of this book. The “third
generation” of IVIGs was made intravenously tolerable by gentle polishing steps
(level D, Fig. 12.1).
Although in many aspects excellent, the two cold-ethanol fractionation methods
reach their limits when pushing for improved recovery without loss of purity. For
this reason, the plasma industry is moving toward the ion-exchange chromatogra-
phy fractionation of IgG. This manufacturing method allows relative high recover-
ies at high purity. Before cryo-poor plasma or the intermediate fraction deprived of
albumin can be fed onto ion-exchange columns, they must be free of lipids (level B,
Fig. 12.1). The most widely used technique to get rid of lipids is by octanoic acid
(=caprylic acid) precipitation. Caprylic acid precipitation was the first to be used in
combination with DEAE cellulose columns for the isolation of IgG from plasma at
good yield (Steinbuch and Audran 1969).
During chromatographic fractionation, lipid-free plasma undergoes several
anion- and/or cation-exchange chromatography steps (Bertolini 1998; Dhainaut
et al. 2013; Cramer et al. 2009; Trejo et al. 2003). The resulting IgG solutions again
undergo polishing steps in order to achieve tolerability (Table 12.5).
With some chromatographically obtained preparations, an elevated frequency
of hemolytic reactions emerged. These adverse events were supposed being
related to the presence of higher titers of isoagglutinins than seen in products
obtained with the cold-ethanol fractionation. In order to reduce levels of isoag-
glutinins anti-A and anti-B, a particular polishing step was introduced by some
manufacturers: immunoaffinity chromatography using columns with corre-
sponding trisaccharides coupled to the matrix (Dhainaut et al. 2013; Höfferer
et al. 2015; Späth et al. 2015).
12.5 W
idening the Pathogen Safety Margins During
Fractionation and Polishing
Donor selection, mass screening, and implementation of added voluntary quality

measures are important for pathogen safety of a plasma product. However, by far
they are not sufficient for an optimal safety bill. “Building in” safety to the fraction-
ation process is what finally can render a product pathogen safe.
Among the stable plasma products, the manufacturing of IgG concentrates
offers the most steps effective enough to reduce or inactivate viruses without
harming too much the biologic function of the molecules. Indeed, IgG concen-
trates largely remained free from transmitting viruses. This was remarkable as
patients with impaired immune defense may require monthly administration of
immunoglobulins lifelong, or patients with autoimmune/inflammatory diseases
might require high to very high doses of immunoglobulin therapy sometimes
over a prolonged period and nevertheless remained virus free. Beside donor
selection and mass screening the third pillar on which pathogen safety is built
on is the reduction of potentially present pathogens during manufacturing (lev-
els B and C, Fig. 12.1). Either an existing fractionation step is validated accord-
ingly, or dedicated virus inactivation/removal steps are introduced to the
manufacturing process. Aspects of the validation process are outlined in
Table 12.6.
There are three, in their principles different, mechanisms that contribute to the
elimination or inactivation of viruses potentially present during manufacturing of
plasma products (Kempf et al. 2007), namely:
162 P.J. Späth
Table 12.6 The essentials of validation of viruses inactivation/removal

A. Downscaling
• Validation studies are performed on a laboratory scale and this implements
downscaling the fractionation process
• The lab-scale process has to undergo validation by demonstrating that critical
parameters of that step correspond in large-scale and laboratory conditions
B. Virus stocks (Table 12.7)
•M odel viruses should be selected for their biologic and physiologic similarities to
plasma-borne viruses; they should be well characterized in size, purity, and aggregate
level; the term model virus defines that all viruses used for validation studies are grown
in cell cultures, event those being human pathogen
• For safety reasons, the use of human pathogen viruses should largely be avoided
•M ust cover a wide range of physicochemical properties of viruses in an effort to test
the ability of the system to cope with pathogen diversity, e.g., enveloped, non-
enveloped, various size, DNA and RNA viruses, and viruses with high resistance to
physicochemical treatment
• Animal viruses in their physicochemical properties should closely resemble the human
pathogen viruses of interest and should represent a range of physicochemical
properties as wide as ever possible
• The virus stock should be of high titer
• When validating IgG manufacturing processes, cross-reacting antibodies to the virus
used should be always considered
C. Cell cultures for read out
• The assay system should be convenient, i.e., cell cultivation should be easy, sensitivity
to virus should be high, and the read out system should be convenient for (electronic)
documentation
• Cell cultures are best prepared in micro titer plates
• Staining of cells not destroyed by virus
D. Validation of a partitioning or filtration step
• The starting material is spiked by a corresponding virus, and the spiked material is
assessed for infectivity, i.e., spiked material is diluted by tenfold (log10 dilution steps)
and is titrated for the tissue culture infectious dose 50% (TCID50)
• The resulting materials (e.g., precipitate and supernatant or filtrate and retained
material/solution) are separately tested for residual infectivity as descried above
• The logarithmic reduction factor (LRF) for a virus is calculated as follows: log10
inoculated virus – log10 residual virus in, e.g., supernatant
• The sum of residual virus infectivity found in, e.g., supernatant and precipitate, must
equal the titer of inoculation; if this is not the case, either an explanation has to be
found and the explanation itself has to be validated or the validation failed
E. Validation of a virus inactivation step
• The inoculated material is incubated while changing an appropriate parameter such as
temperature, and aliquots are taken at various incubation times
•E ach aliquot is assessed for remaining infectivity, and the kinetics of virus inactivation
is calculated
• The appropriateness of results is proven by calculation of inactivation constants,
transformation into natural logarithms, and plotting against the reciprocal of absolute
temperature (Arrhenius plot). The fitting of calculated values on a regression line is an
excellent internal control for quality of data
Table 12.7 Human viruses and their model virusesa recently used for validation studies
Human pathogen Model virus Size of the viruses (nm)
B19 virus (Parvoviridae) Minute virus of mice (MVM); 18–25
canine parvovirus (CPV)
Hepatitis A, poliovirus Encephalomyocarditis virus 30
(Picornaviridae) (EMCV)
Hepatitis B virus (enveloped Infectious bovine rhinotracheitis 200
DNA viruses) virus (IBRV)
Hepatitis C, West Nile Bovine viral diarrhea virus 40–60
(Flaviviridae) (BVDV); WNVb; Sindbis virus 70
(SINV)
Human immunodeficiency HIVb 80–100
virus (Retroviridae)
Herpes 1 and 2, HHV-8 Pseudorabies virus (PRV) 100–180
(Herpesviridae)
Vaccinia, cowpox, smallpox Approx. 250 and higher
(Poxviridae)
TSE agent (PrPSc) 1 IU is assumed to
contain ≥20 aggregated
proteins having a
calculated size of
16–56 nm
a
Viruses used in validation studies should be well characterized in size, represent as closely as pos-
sible viruses which might be present in plasma, cover a wide range of physicochemical properties
of viruses, e.g., enveloped, non-enveloped, various size, DNA and RNA viruses, and viruses with
high resistance to physicochemical treatment. In validation studies with immunoglobulins, care
has to be taken that results are not misleading due to the presence of cross-reactive and/or neutral-
izing antibodies
b
Although human pathogen, viruses grown in cell cultures are all considered “model”
1. Inactivation
• Solvent/detergent (S/D) treatment: disruption of virus envelope
• Caprylic (octanoic) acid treatment
• Treatment a low pH: destructive conformational changes of structural
proteins
• Heat: dry or wet (wet = pasteurization), disruption of envelope and destruc-
tive conformational change of structural proteins (e.g. capsid proteins)
2. Elimination based on size: virus filtration (formerly termed nanofiltration).

Elimination at large scale of possibly contaminating pathogens based on size is
the most recent technique to free plasma products form pathogens. Virus filtra-
tion at large scale was introduced by the Central Laboratory of the Swiss Red
Cross, Blood Transfusion Service in the late 1990s (virus filtration on a modest
size (Stucki et al. 1997); virus filtration at large scale: submission dossier for
Sandoglobulin NF in 1999). With the progress in manufacturing of filters with
smaller and smaller pores, virus filtration has become a universal key process in
assuring pathogen safety—size matters only.
164 P.J. Späth
3. Partitioning, e.g., virus in the precipitate while the supernatant, is processed

further. Filtration with filter aids can take advantage of the filter aids tightly
binding and adsorbing viruses in precipitates and despite their small size retain-
ing them (technical term: depth filtration). Partitioning involving filtration tech-
niques are not to confound with virus filtration that is a dedicated virus removal
principle.
The highest level of product safety is achieved when all three principles are
applied in a stepwise and diligent manner during the fractionation process
(Table 12.8). As an example the LRFs obtained by various methods are listed in
Table 12.9. LFRs obtained on basis of varying principles are additive in defining the
safety margin of an entire manufacturing process.
Table 12.8 Aspects of dedicated virus inactivation/removal processes

S/D Low pH Heat/pasteurization Virus filtration
Enveloped viruses +a + + +
Non-enveloped viruses − ± ±/(−) +
Prions − − − +
Except on Poxviridae
a
Table 12.9 Examples are depicted of margins of pathogen inactivation and removal during frac-
tionation of IVIG staring from possibly contaminated starting material
Principal mechanism and methods of pathogen
reduction HIV HAV HCV HHV B19V
Partitioning (separation into different physical phases)
Depth filtration with filter aids (A) 11.5 4.6–10.2 12.4
Depth filtration with filter aids (B) ≥5.3 4.2 2.1 ≥6.3 2.3
Ion-exchange chromatography (1)
Ion-exchange chromatography (2) ≥3.0 ≥1.4 4.0 ± 0.3 ≥3.3
Inactivation by alteration of structures which are essential for the infectivity of the pathogen
Pasteurization (wet heat) ≥5.5 ≥5.4 ≥5.7
Low pH ≥5.4 4.6 ≥5.9 >3
Low pH and traces of pepsin 6.1 <1.0 >4.4 and >5.3 n.a.
>6.7
Solvent/detergent treatment ≥6.0 n.a. ≥7.8 ≥8.4 n.a.
Caprylate incubation ≥4.5 n.a. ≥4.5 ≥4.6 n.a.
Combination of partitioning and inactivation 4.0 3.4 1.4–3.6 3.6
Elimination based on size
Virus filtration A ≥5.3 >5.1 >5.1 and
>7.5
Virus filtration B ≥5.3 ≥3.7 ≥2.7 ≥5.5
Numbers indicate logarithmic reduction factors (LRFs). Examples are taken from literature and
package inserts. The human pathogen viruses aimed to eliminate or inactivate are indicated on
column heads. The effectively used model viruses are given in Table 12.6
The results published by various manufacturers of the various virus inactivation

and removal steps allow to conclude that by applying the various techniques
sequentially:
• Inactivation of enveloped viruses is usually achieved well.

• Current inactivation methods show limited efficiency for non-enveloped viruses.
• Virus filtration has the potential to completely remove the smallest and most
robust non-enveloped viruses known.
• Partitioning processes additionally contribute to viral safety.
The combination of these pathogens elimination methods also provides confi-

dence that the various fractionation processes can cope with newly emerging
viruses.
12.6 Emerging Human Pathogens
So far, no proven transmission by IgG concentrates of (re-)emerging viruses in the

last two decades occurred. This might be credited to the tightly implemented QA
framework. The (re-)emerging pathogens are predominantly of zoonotic nature.
Hence, their reservoirs are animals and their (re-)emergence is associated with
hygienic conditions. Transmission of enveloped zoonotic viruses over the species
barrier with severe consequences for man have been described for Chikungunya
virus, MERS coronavirus, avian influenza virus (bird flu; H5N1), SARS coronavi-
rus, West Nile virus, Ebola virus, Zika virus, others. The presence of these viruses
in blood donors were reported for West Nile virus, SARS coronavirus and Zika virus
which persists in the cellular blood compartment. Validation studies have assured
the safety regarding transmission by plasma products of relevant enveloped zoo-
notic viruses (Table 12.10). In general, enveloped viruses can relatively easily be
inactivated, e.g., by solvent/detergent treatment.
For non-enveloped viruses, gene mutations are a prerequisite to cross the species
barrier. Hepatitis E virus (HEV) is a non-enveloped zoonotic virus of pigs. Four
genotypes are known. Genotypes 3 and 4 are recognized as zoonotic pathogens in
industrialized countries. Transmissions are by pork blood (in rare cooked meat).
Transmission of HEV by plasma exchange has been reported (Mallet et al. 2016).
Prevention of HEV transmission is relevant as infection can have severe neurologi-
cal consequences, even in immunocompetent individuals (Blasco-Perrin et al. 2015;
Higuchi et al. 2015; Pérez Torre et al. 2015; Scanvion et al. 2017). Elimination or
inactivation of HEV during the manufacturing of plasma products apparently is suf-
ficient in preventing pathogen transmission (Table 12.10).
In general non-enveloped viruses do mutate slowly, with one known exception,
the DNA of Parvoviridae which shows a similar mutation frequency to RNA viruses
(Boschetti et al. 2005; Shackelton and Holmes 2006). Under distinct immune and
166 P.J. Späth
Table 12.10 Emerging zoonotic human pathogen viruses and their model viruses used for valida-
tion studies
Size of the
viruses Validation
Human pathogen Model virus (nm) studiesa
Chikungunya virus (CHIKV, Bovine viral diarrhea 40–60 Leydold et al.
enveloped; Flaviviridae) virus (BVDV) (2012)c
Sindbis virus (SINV)b 50–70
CHIKV strain
“LR2006-OPY1”
Hepatitis E virus (HEV, non- Feline calicivirus (FCV) 32–34 Farcet et al.
enveloped; Herpesviridae) (2016)
Severe acute respiratory syndrome- Frankfurt-1 strain of Yunoki et al.
associated coronavirus (SARS-CoV, SARS-CoV (2004)
enveloped) SARS-CoV isolate Rabenau et al.
FFM-1 (2005)
West Nile virus (WNV, enveloped; Bovine viral diarrhea 40–60 Kreil et al.
Flaviviridae) virus (BVDV) (2003)
Zika virus (ZIKV, enveloped; ZIKV strain 40–60 Blümel et al.
Flaviviridae) PF13/251013-18 (2017)
ZIKV isolate H/ 40–60 Kühnel et al.
PF/2013 (2017)
Except for HEV, all are enveloped viruses
a
The logarithmic reduction factors (LRFs) are given in the publications.
b
A model virus of the early days of validation studies
c
This study provides solid reassurance for the safety of plasma products in regard to emerging
viruses. Furthermore, the results verify that the use of model viruses is appropriate to predict the
inactivation characteristics of newly emerging viruses when their physicochemical properties are
well characterized
adaptive pressures, specific amino acid changes in parvoviruses endowed tropism

shifts in the animal world (Lopez-Bueno et al. 2006). Hence, new variants/species
tropisms have to be expected. Theoretically, small non-enveloped viruses represent
the biggest threat for transfusion incidences in the future, and hopefully the tech-
nique of virus filtration is sufficient or will be improved to eliminate eventually
emerging Parvoviridae with human tropism.
12.7 Transmissible Spongiform Encephalopathies (TSEs)
Also of animal origin is the agent of the variant Creutzfeldt-Jakob disease (vCJD), a
TSE. vCJD with great certainty was transmitted from cow (mad cow disease or bovine
spongiform encephalitis = BSE) to humans (Bruce et al. 1997). BSE emerged due to
an “optimized” production process of animal carcass-derived material fed to cows
(Wilesmith et al. 1991). Other forms of human spongiform encephalitides exist and
can be iatrogenic/sporadic, inherited/genetic, or acquired/infectious (Table 12.11).
Spongiform encephalitides are mediated by the misfolded isoform “scrapie” of a
normal cellular prion protein, PrPC which is ubiquitous in cells and is a protein
highly conserved over the evolution. The misfolded “scrapie” prion protein (PrPSc)
results in fatal neurodegenerative diseases of mammals. Although a protein-only

agent, PrPSc is “infectious” in a sense that the misfolded protein might induce con-
formation changes of the normal PrPC resulting in sticky rod-like and fibrillary par-
ticles accumulating in the brain.
Serum, plasma, and leukocytes from vCJD patients might harbor infectious
PrPSc. Indeed, transmission of PrPSc has been reported in three symptomatic and one
non-symptomatic cases of non-leukocyte-depleted red blood cell concentrates
(Hewitt et al. 2006; Peden et al. 2004). Measures to eliminate possibly contaminat-
ing PrPSc during plasma fractionation became mandatory. It is accepted that inacti-
vation of PrPSc is not possible without destruction of the biological activity of a
plasma product. Hence, elimination by partitioning or exclusion by size remains the
two possibilities (Cai et al. 2002). Validation studies were performed at different
manufacturers’ site with different spikes and different detection systems. The manu-
facturing steps studied for removal capacity of PrPSc included precipitation, adsorp-
tion, chromatography, and filtration, as well as combined steps. They proved to be
highly potent in removal of the TSE agents possibly contaminating plasma (Cai
et al. 2013). Reduction by individual steps in sequence was additive (Table 12.12).
Table 12.11 Spongiform encephalopathies (SEs) of mammals
Animals Humans
Kuru (cannibalism; acquired/infectious)
Sheep → Scrapie
Gerstmann-Sträussler-Scheinker-
Deer & Elk → Chronica Syndrome (GSS; inherited)
wasting disease (CWD)
Fatal familial insomnia (FFI; inherted)
Mink → MSE Creutzfeldt-jakob Disease; classical =
idiopathic/sporadic form (CJD or
Cat → FSE cCJD/sCJD)
pathogenic prion protein almost
exclusively located in the CNS
Live stock carcass derived
material fed to ruminants Creutzfeldt-jakob Disease; new variant
form (vCJD; acquired/infectious)
Bovine → BSE
pathogenic prion protein found in
CNS but also in abundance in some
lymphatic tissue
Table 12.12 Elimination of PrPSc during manufacturing of IgG concentrates

Principal mechanism and methods of pathogen reduction PrPSc
Partitioning (separation into different physical phases)
Depth filtration with filter aids 7.3
Ion-exchange chromatography ≥5.4
Elimination based on size
Virus filtration (A) 4.4
Virus filtration (B) ≥4.3
Logarithmic reduction factors (LRFs) are given. Inactivation was never assessed because not appli-
cable (see text)
168 P.J. Späth
Furthermore, the declining incidence of vCJD, the stringent donor selection due to
geographic donor deferral policy, and the additive nature of removal steps render the
agent of vCJD unlikely to be transmitted by plasma products. Indeed, no proven
transmission of vCJD by IgG concentrates was ever reported (Ritchie et al. 2016),
even not when the administration of a possibly PrPSc contaminated IgG concentrate
occurred (El-Shanawany et al. 2009). Ascertained transmission by other plasma
products has not been described neither.
The sporadic/iatrogenic CJD (sCJD) is not the same as vCJD (Table 12.11).
sCJD represents about 85% of all spongiform encephalitides of humans and affects
elderly people. There exist several surveillance studies on the transmission of sCJD
by blood and blood products. These are conducted by the American Red Cross, in
the UK and in France (Crowder et al. 2017; Martin and Trouvin 2013; Urwin et al.
2016). These studies did not report sCJD in patient populations treated with blood
and plasma products. This was the basis not to consider sCJD transmissible by
blood transfusion or by plasma products. However, the Emerging Infectious Disease
Journal in May 2017 posted in electronic form a report on two patients receiving
coagulation factor concentrates and dying from sCJD. Authors conclude (citation):
“A causal link between the treatment with plasma products and the development of
sCJD has not been established, and the occurrence of these cases may simply reflect
a chance event in the context of systematic surveillance for CJD in large popula-
tions.” It will be of outmost importance whether and how many new cases will be
reported in future.
12.8 C
ompleting the Full Package for a Safe and Efficacious
Immunoglobulin Concentrate
Final formulation: To ensure stability/tolerability of a product over its shelf life, the
right selection of stabilizers is of particular importance. Products can by lyophilized
(=freeze-dried). The basic physicochemical process of freeze-drying is the replace-
ment of the layer of water surrounding the IgG molecules. This layer, which is also
termed “hydration shell” or “hydration sphere,” keeps the IgG molecules in solution
(Makarov et al. 2002). In dehydrated products, e.g., oxidation of the preparation is
largely prevented, and aggregation or dimer formation is suppressed. Hence, the
excipient only to a part serves stabilization of a freeze-dried product, while its major
role is to make sure the lyophilized protein can be reconstituted entirely within rea-
sonable time (no aggregates remaining). The compounds that can be injected intra-
venously with the best physicochemical properties to simulate water are sugars
followed by some amino acids. Although associated with osmotic nephrosis in
patients at risk, several lyophilized products containing sucrose are still on some
markets (see also Chap. 13).
In response for the request of more convenient handling, the liquid preparations
were developed. In liquid preparations, aging and continuous interaction of mole-
cules are inherent. The stabilizer has to preserve the characteristics without hamper-
ing their quality by preventing oxidation and IgG-IgG interactions, e.g., limiting
IgG dimer formation. The most widely used stabilizers for liquid preparations are
glycine, L-proline, maltose, and sorbitol.
Cleaning and sanitation of a production line: Prevention of cross-contamination
through surface-adsorbed infectious agent is a measure to be taken under
cGMP. Cleaning validations involving infectious agents are problematic, e.g.,
because the cleaning process has to be scaled down. This in most cases is impossi-
ble, e.g., because the rheological properties are different on a small scale. Typically
NaOH or sodium hypochlorite (NaClO) are used for plant sanitation after a run and
at appropriate concentrations are sufficient to destroy virus infectivity. However,
how about the TSE agent of vCJD? It needed a particular effort to show that there
are sanitation measures that can destroy the TSE agent PrPSc. PrPres served this pur-
pose (Table 12.13). The validation strategy was to show that PrPres after NaOH/
NaClO exposure becomes on one hand noninfectious and on the other hand sensi-
tive to proteinase K digestion. NaOH and NaClO treatment both destroy PrPres even
at low NaOH and NaClO concentrations. Regulatory agencies consider current
cleaning regimes adequate to assure batch-to-batch segregation.
Traceability: GMP requirements for blood and plasma derivatives include the
traceability of batches from donor to recipient. The industry is responsible for inter-
linking a given batch of plasma product with information such as responses of the
donor has given on the medical questionnaire, results of mass screening and pooling
information. Traceability also has to interconnect batches and the hospital where the
product was delivered. It is the responsibility of the hospital to complete traceability
by documenting which IVIG batch has been given to which individual patient(s).
Table 12.13 Some physicochemical properties of prion proteins of mammals in health and
diseasea
Condition/prion protein
Origin (agent) Some properties
Mammals Health/PrPC An ubiquitous, normal, native, noninfectious
protein of cells encoded on the short arm of
chromosome 20
No tendency to aggregate
Complete degradation by proteinase K
Mammals Spongiform encephalitides/ Aberrantly folded prion protein
PrPSc Tendency to convert PrPC into the misfolded
form thereby aggregating and forming
filaments and plaques that destroy CNS cells
Only partially digested by proteinase K
Detection by Western-blotting indicates
infection of the tissue examined
Various strains known
Artificial No biological function/PrPres Fragment of PrPSc resistant to digestion upon
prolonged incubation with proteinase K;
PrPSc ≠ PrPres
a
General term of disease = transmissible spongiform encephalopathy (TSE) mediated by PrPSc;
PrPSc is a generic term for aberrantly folded, “infectious” prion proteins and does not refer exclu-
sively to the suspected agent of scrapie of sheep
170 P.J. Späth
This enables “look backs” in case any problem should be identified at the donor or
recipient end. The basis of the traceability is a bar code identification system. A
proper traceability system ensures that each lot can be recalled, should this be nec-
essary. It further allows the handling of post-donation information and pharmaco-
vigilance in an efficient and safe manner.
Obtaining the final proof of clinical efficacy and pathogen safety of an IgG con-
centrate: Quality assurance through surveillance programs extends product quality
after its distribution. Pharmacovigilance, post-marketing studies and surveillance
programs are the pillars on which the final proof for the quality of an IgG concen-
trate stands. Pharmacovigilance ensures the continued safety of medicinal products
in use by collecting, monitoring, and assessing any type of adverse drug reactions
related to the product (Ball et al. 2016), http://ec.europa.eu/health/human-use/phar-
macovigilance_en. For some infections, incubation time might be long. Surveillance
programs are currently the only means to conclusively obtain long-ranging safety
information. Clinicians are encouraged to report adverse events, especially those
that are unexpected or unusual.
Acknowledgments The help of Christoph Kempf, University of Berne, is greatly appreciated, as

well as information kindly provided by Roland Hubner, The Federal Public Service (FPS) Health,
Food Chain and Environment, Belgium.
Some web sites for additional information:
http://www.cjd.ed.ac.uk
http://www.eurocjd.ed.ac.uk
http://www.who.int/home-page/
http://www.ukhcdo.org/patient-information
http://case.edu/med/pathology/centers/npdpsc
http://www.who.int/medicines/areas/quality_safety/regulation_legislation/ListMRAWebsites.pdf
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Current IgG Products and Future
Perspectives 13
Peter J. Späth
13.1 Introduction
Most international IG products are FDA and/or EMA approved. For some countries,
some IG products might have formulation characteristics that allow their safe use
although the supply chain might be less well standardized as in others. In case a
country might not be willing to build up the complex organization necessary for
plasma fractionation, an established fractionation company might be willing to offer
a fractionation technique they formerly have used. In such a case, the country
designs a dedicated organization that is responsible for plasma collection under
supervision of authorities, and the collected plasma is shipped to a fractionating
company for fractionation. The final product is shipped back to the country and is
distributed to hospitals. The entire process is termed “toll manufacturing.”
Progress in IG manufacturing and formulations always are driven by enhancing
recovery without losing in purity. Liquid formulation and storage at room tempera-
ture are important for hospital pharmacies and bedside activities. Tolerability at
high infusion rates/high strength of the IG solutions is a hospital and medical prefer-
ence. Subcutaneous application first reported in 1952 (Bruton 1952) and not prac-
ticed for the next 30 years (Berger et al. 1980) has meanwhile enhanced convenience
and partly shifted the IG administration from hospital to home.
The tables intend giving an overview and serve as an orientation. The tables were
generated from summaries of product characteristics, prescribing information, form
package inserts, websites of regulatory agencies, and the literature. It has to be
stressed that the tables do not replace a package insert and that they should not serve
a basis for therapies.
Excluded from the tables are the maximal infusion rates of the different products,
which are the main source for adverse events (Späth et al. 2015).
P.J. Späth

176 P.J. Späth
Availability of IG products: It is the great merit of the International Patient

Organization for Primary Immunodeficiencies (IPOPI) updating accurately and con-
tinuously the availability of immunoglobulin concentrates around the world. It is
possible to look up which products companies provide or which products are avail-
able in a given country (http://www.ipopi.org/index.php?page=immunoglobulin-
countries; accessed July 2017). For particular countries/markets or IgG concentrates,
it is further recommended to search on the website of the WHO mentioning all local
registration agencies around the world http://www.who.int/medicines/areas/quality_
safety/regulation_legislation/ListMRAWebsites.pdf (accessed July 2017).
13.2 Update on Available IgG Concentrates
Tables below provide the update on available IgG concentrates. The tables focus on
the characteristics and contents of the IG preparations.
The first Table 13.1 lists the products obtained by processes mainly using ion-
exchange chromatography. The roots of isolation of IgG from human plasma by
ion-exchange chromatography go back to the late 1950s (Fahey and Horbett 1959).
The first industrial applications were small-scale isolations using DEAE-sepharose
(Baumstark et al. 1964; Bowman et al. 1980; Hoppe et al. 1973). The chromato-
graphic method allowed obtaining products for intravenous use at relative high
recovery (Friesen et al. 1985).
Table 13.2 lists products fractionated with the “cold ethanol” techniques to which
the most recent improvements have been added.
Table 13.3 depicts products with which many important clinical breakthroughs
were achieved and which represent the fundament for the products in Table 13.2.
Table 13.4 summarizes the subcutaneously applicable IG products for replace-
ment therapy and for immunomodulation in chronic diseases.
Table 13.5 lists hyp erimmune globulins fractionated from plasma of selected
(convalescent) donors or donations having high titers in a given antibody
specificity.
Table 13.6 lists some “domestic” IgG concentrates.
Additional information regarding some of the products mentioned

Several plasma fractionators have a strong commitment for toll manufacturing.
A contract for toll manufacturing includes delivery of plasma collected by, e.g.,
national services, the processing of the plasma, and shipping back the final product.
As an example, plasma collected in Australia, New Zealand, Hong Kong, Singapore,
Malaysia, and Taiwan are processed according the Intragam P method and are dis-
tributed in their countries of plasma origin under the brand name Intragam P or
other. LFB has similar contracts for some North African countries.
Intragam P was the first very large-scale produced IgG concentrate applying the
chromatographic technique.
Sandoglobulin: was the first product on the market composed of “native” IgG
molecules. Originally, the product was termed “IgG-SRK” (SRK = Swiss Red
Cross) (Schnoz et al. 1979). IgG-SRK was the product with which the
Table 13.1 Immunoglobulin concentrates manufactured using the ion-exchange chromatography technique as main fractionation method$#
Strength
IgG concentrate of the
(manufacturer, IgG solution
financial Polishing and validated cont. ready to Storage Carbohydrate Sodium IgA cont.
background) pathogen reduction Formulation (%) use (%) conditions Osmolality stabilizers cont. pH (mg/mL)
ClairYg® (LFB Initial cold ethanol Mannitol, NR 5 2–8 °C for 270– None None 4.8 ± 0.1 ≤0.022
Biomedicaments, step, S/D treatment, glycine, 2 years (RTa 330 mOsmol/kg
LFB SA, France) virus filtration polysorbate 80 for 1 year)
Gammaked™ Gammaked™ is identical to Gamunex
(Grifols USA,
Kedrion Biopharma,
S.p.A, Italy)
Gamunex® (Grifols Initial cold ethanol Glycine ≥98 10 +2 °C to 258 mOsmol/kg None None 4.0–4.5 ≤0.046
USA Llc, Grifols step; caprylate 0.16–0.24 M +8 °C solvent added
13 Current IgG Products and Future Perspectives
SA, Spain) precipitation and (refrigerator)

filtration; anion- for 3 years;
exchange ≤6 months at
chromatography; RT with shelf
ultrafiltration; low pH life ending at
this 6-month
period
Hizentra® (CSL Hizentra® is registered for subcutaneous application (see there); beside the strength of the solution (20%), manufacturing corresponds to
Behring, CSL Ltd., Privigen®
Australia)
(continued)
177
178
Strength
Intragam P® b (CSL Initial cold ethanol Liquid maltose, ≥98 6 2 °C–8 °C Isotone Maltose NR 4.25 ≤0.025
Behring, CSL Ltd., step, delipidation 0.1 g/mL (10% (refrigerator);
Australia) pasteurization, low pH w/v) or store below
25 °C and use
within
3 months
Iqyumune® (LFB Initial cold ethanol Liquid, ≥95 10 24 months, 281 mOsmol/kg None None 4.6–5.0 ≤0.028
Biomedicaments, step; caprylic acid glycine, RT added
LFB SA, France) precipitation; polysorbate 80
immunoadsorption
chromatography, S/D
treatment; virus
filtration, ultrafiltration
Privigen® (CSL Initial cold ethanol L-proline ≥98 10 4.8 mL/kg/h 320 mOsmol / None Traces 4.6–5.0 ≤ 0.025
Behring, CSL Ltd., step; octanoic acid 210– (EU) 0.04 mL/ kg
Australia) precipitation; pH 4; 290 mmol/L kg/min in
virus filtration; depth chronic ITP
filtration; and 0.08 mL/
immunoaffinity kg/min in PID
chromatography (US)
P.J. Späth
Strength
Rhophylac® (CSL Rhophylac is a polyclonal hyperimmune anti-blood group Rh(D) IgG concentrate; for details see Table 13.5
Behring, CSL Ltd.,
Australia)
Tegeline New® Tegeline New is identical to ClairYg and is distributed e.g. in Brazil (http://www.lfb.com.br/atualidades/
(LFB nova-imunoglobulina-intravenosa-liquida-do-grupo-lfb-tegeline-newy/
Biomedicaments,
LFB SA, France)
$
All preparations listed are liquid formulations
#
Main fractionation by ion-exchange chromatography for most products include pre-purification by cryo-precipitation and one cold ethanol step to separate
from the starting material for albumin fractionation (see Chap. 12). This forefront technique allows high recoveries at high puritiesc
cont = content, max = maximum, NR = not reported, w/w = weight/weight, CSL Commonwealth Serum Laboratories, LFB Laboratoire Français du Fractionnement
et des Biotechnologies
a
RT = room temperature, i.e., not over 25 °C
b
Intragam P was the first large-scale chromatographically purified IgG concentrate. The author knows about earlier attempts for chromatographic purification
in Humgary (Human, Gödöllő) using a Pharmacia chromatographic system
c
The first mainly chromatographically obtained products were WinRho® SDF and Varizig® (Aptevo BioTherapeutics, Emergent BioSolutions, USA).
Manifacturing used DEAE-Sephadex a method no more the most forefront chromatographic technique. WinRho SDF and Varizig are polyclonal hyperimmune
anti-blood group Rh(D) and anti-varizella IgG concentrates, respectively- For details, please consult Table 13.5
179
180
Table 13.2 Newer IgG concentrates obtained by the cold ethanol fractionation techniques according to Cohn and Onlcey (C&O) or Kistler and
Nitschmann (K-N)
Strength
of the
protein
IgG concentrate solution
(manufacturer, Type of cold Polishing and IgG ready for
organization, ethanol validated pathogen cont. infusion IgA cont.
country) fractionation reduction Excipient (%) (%) Osmolality Sugars Sodium cont. pH (mg/mL)
Line extensions of well introduced liquid IVIGs as indicated in Table 13.3
Flebogammadif® C&O PEG precipitation; D-sorbitol 5% ≥97 10 240– None <3.2 mmol/L 5.0– ≤0.006
10% DIF (Grifols, ion-exchange 370 mOsmol/L 6.0
Grifols SA, Spain) chromatography;
10% low pH;
pasteurization;
solvent/detergent;
virus filtration
Gammagard® C&O Cation- and Glycine 250 mmol/L ≥98 10 240– None No added 4.6– ≤0.037
liquid (Baxalta, anion-exchange 300 mOsmol/kg sodium 5.1
Shire plc, Jersey) chromatography;
low pH; S/D; virus
filtration
Gammaplex® 10% C&O Ion-exchange Glycine200–300 mM, ≥98 10 Typically None ≤30 mM 4.9– ≤0.02
(BPL, Creat Group chromatography, polysorbate 80 280 mOsmol/kg 5.2
Corp., China)a S/D treatment, virus 1–6 mg
filtration, low pH
P.J. Späth
Strength
of the
protein
Intragam P 10% C&O Low pH, Glycine NR 10 350 mOsmol/kg None NR 4.25 Typically
(CSL Behring, pasteurization 2.25 g/100 mL <0.025
CSL Ltd.,
Australia)
Intratect® 10% C&O Octanoic acid/ Glycine 300 mMol/L ≥96 10 300 mOsmol/kg None <10 mmol/L NR ≤1.8
(Biotest pharma, calcium acetate;
Biotest AG, cation-exchange
Germany)a chromatography;
solvent/detergent;
ultra and diafiltration
Kiovig® (Baxalta, Kiovig® corresponds in its composition to Gammagard liquid, except for the IgA content which is ≤0.14 mg/mL; Kiovig® is the trade name for
Shire plc, Jersey) the EU market; see further details at Gammagard liquid
Octagam® 10%a C&O Chromatography; Maltose 90 mg/mL ≥95 10 ≥240 mOsmol/ Maltoe ≤30 mmol/L 4.5– ≤0.400
(Octapharama, pH 4; solvent/ kg 5.0
Octapharma AG detergent;
Switzerland) ultrafiltration
Panzyga® K-N Ion-exchange Glycine ≥95 10 ≥240 mOsmol/ None <0.03 mmol 4.5– <0.3
(Octapharma, chromatography S/D 17.0 ± 0.29 mg/mL kg (or 0.69 mg)/ 5.0
Octapharama, AG, treatment 20 nm mL
Switzerland) virus filtration
(continued)
181
182
Strength
of the
protein
Recent IVIGs with new formulations
Bivigam™ (Biotest C&O Partitioning, S/D 0.20–0.29 M glycine, ≥96 10 NR None 0.100–0.140 M 4.0– ≤0.2
pharma, Biotest treatment, 35 nm 0.15–0.25% 4.6
AG, Germany)b virus filtration polysorbate 80
Vigam® (BPL, C&O Cation-exchange Human albumin 20% ≥95 5 NR Sucrose Sodium added 4.8– <0.014
Creat Group Corp., chromatography; Glycine added 5.1
China) low pH; S/D Sucrose
In most cases, 5% preparations were developed further to obtain a stable, safe and clinically well-tolerated solution of 10% IgG. Products intended for intrave-
nous use are listed
C&O: (Cohn et al. 1944; Oncley et al. 1949); K-N: (Nitschmann et al. 1954)
BPL Bio Products Laboratory, CSL Commonwealth Serum Laboratories
a
In some countries several distributors for the product might exist
b
Distribution in the USA of Bivigam™ by Kedrion US terminated as of January 2017: https://www.kedrion.us/bivigam%C2%AE-will-no-longer-be-avail
able-sale-or-distribution-2017
P.J. Späth
Table 13.3 IgG preparations for i.v. use available for decades
Strength
of the
Type of protein
cold Polishing and solution
IgG concentrate ethanol validated IgG ready for
(manufacturer, fractio- pathogen Excipients cont. infusion Sugars as IgA cont.
organization, country) nation reduction stabilizer (%) (%) Osmolality excipients Sodium cont. pH (mg/mL)
Liquid polyclonal IgG concentrates registered for intravenous application
Flebogamma® 5% DIF C&O PEG D-sorbitol 5% ≥97 5 240–350 mOsmol/L None <3.2 mEq/L 5.0–6.0 ≤0.05
(Grifols, Grifols SA, precipitation;
Spain) ion-exchange
chromatography;
pasteurization
Gammaplex® 5% (BPL, C&O Ion-exchange D-sorbitol ≥95 5 NR None <0.85% 4.9 ≤0.01
Creat Group Corp., China) chromatography, Glycine
S/D treatment, Polysorbat 80
virus filtration,
low pH 2–25 °C
for 24 months
IG Vena N (Kedrion, C&O Low pH; S/D Maltose 10% ≥95 5 NR Maltose 3 mmol/L NR <0.05
Kedrion Biopharma
S.p.A. Italy)
Gammaplex® 5% (BPL, C&O Ion-exchange D-sorbitol ≥98 5 NR Sorbitol Yes, NR 4.8–5.1 ≤0.01
Creat Group Corp., China) chromatography, (max 55 mg/
S/D treatment, mL), glycine,
virus filtration, polysorbate
low pH 80
Sodium
acetate
0.52 mmol/L
183
(continued)
184
Strength
of the
Type of protein
Intragam P (CSL K-N Low pH, Maltose ≥98 6 NR Maltose NR 4.25 Nominally
Bioplasma, CSL Ltd., pasteurization 10 g/100 mL ≤0.025
Australia)
Intratect 5% (Biotest C&O Glycine ≥96 5 None NR ≤0.9
Pharma; Biotest AG,
Germany)a
Octagam® 5% C&O Chromatography; Maltose ≥95 5 310–380 mOsmol/ Maltose ≤0.015 mmol/L 5.1–6.0 ≤0.2
(Octapharma,Octapharma, pH 4; solvent/ 10 mg/mL kg
AG, Switzerland)a detergent;
ultrafiltration
Nanogam® (Sanquin, K-N pH 4.4/trace Glucose ≥95 5 NR Glucose NR NR ≤0.006
Sanquin Plasma Products pepsin; solvent/ 50 mg/mL
B.V., Netherlands) detergent;
nanofiltration
3 years at
2 °C–8 °C (in a
refrigerator)
Lyophilized polyclonal IgG concentrates for intravenous use after reconstitution
Carimune/Carimune® NF K-N Partitioning; Lyophilized ≥96 3, 6, 9, 192–1074 mOsmol/ Sucrose <20 mg/g of 6.6 720
(CSL Behring, CSL Ltd., pH 4/trace powder 12 kg (depending on protein
Australia) pepsin; the IgG
nanofiltration concentration of the
reconstituted
preparation)
P.J. Späth
Strength
of the
Type of protein
Gammagard S/D 5% C&O DEAE Glucose 20%, ≥90 5 636 mOsmol/L Glucose <8.5 mg/mL 6.8 ± 0.4 <0.001
(Baxalta, Shire plc, Jersey) chromatography glycine 0.3 M
S/D human
albumin 3%
Sandoglobulin®/ Sandoglobulin® NF identical to Carimune® NF; see above
Sandoglobulin® NF (CSL Carimune®/Sandoglobulin® are virus filtered as well
Behring, CSL Ltd.,
Australia)
Tegéline® LFB, LFB SA, K-N pH 4/trace Sucrose 2 g/g >97 5 NR Sucrose 8 mg/10 mL NR 17 mg/g
France) pepsin; filtration; protein protein

virus filtration
A chemically treated liquid IgG concentrate (5%) distributed in several markets (see also Table 13.6)
Pentaglobin a preparation C&O β-propiolacton/ Glucose 5 NR Glucose 78 mmol/L NR Enriched
enriched in IgM and IgA UV treated 25 g/L in IgA
(12% each) (Biotest (and IgM)
Pharma, Biotest AG,
Germany)
Some of these preparations were instrumental in progress of clinical use of IVIGs. Those of the early native, well-tolerated preparations which still are on the
market, all got a brush up in order to comply with viral safety requirementsb. For checking the availability of the concentrate in a given market, the reader is
advised to visit the following website: http://www.who.int/medicines/areas/quality_safety/regulation_legislation/ListMRAWebsites.pdf (accessed April 2017)
BPL Bio Products Laboratory, CSL Commonwealth Serum Laboratories, LFB Laboratoire Français du Fractionnement et des Biotechnologies, cont. = content,
DEAE = diethylaminoethyl, max = maximum, NR = not reported, PEG = polyethylene glycol, w/w = weight/weight
a
In some countries several distributors for the product might exist
b
Upon request, reports on more recent clinical studies with some of these older products are available from the author
185
186
Table 13.4 IgG concentrates intended for s.c. applications

Main fractionation Formulation and IgG Strength Sugar IgA content
Brand name/manufacturer method Polishinga stabilizer content (%) content pH (mg/mL)
Polyclonal IgG concentrates registered for s.c. use only
Beriglobin® P(CSL Cold ethanol The measures taken Liquid glycine ≥98 16 NR NR 0.8–1.6
Behring, CSL Ltd., are considered (contains 100 IU/vial
Australia) effective for antibodies to hepatitis
enveloped and B virus)
non-enveloped viruses
Cuvitru®—IGSC 20 Cold ethanol S/D virus filtration Liquid glycine 0.25 M ≥98 20 None 4.6–5.1 ≤0.08
(Baxalta, Shire plc, Jersey) low pH
Evogam® (CSL Behring, Cold ethanol Pasteurization Liquid ≥98 16 None 5.5 NR
CSL Ltd., Australia) Virus filtration Glycine
2.25 g/100 mL
Hizentra®/IgPro20 (CSL Ion-exchange S/D Liquid ≥98 20 None 4.6–5.2 ≤0.05
Behring, CSL Ltd., chromatography Low pH L-proline 250 mmol/L
Australia) Virus filtration Polysorbate 80
10–30 mg/mL
Gammanorm® Cold ethanol S/D Liquid; glycine, ≥95 16.5 None ≤0.0825
(Octapharma Sweden, polysorbate 80,
Octapharma AG, Refrigerator
Switzerland)
Octanorm® (Octapharma Identical to Gammanorm
Sweden, Octapharma AG,
Switzerland)
P.J. Späth
Subgam® (BPL, Creat Cold ethanol The measures taken Liquid ≥95 16 NR NR <0.04% w/w
Group Corp., China) are considered
effective for
enveloped viruses and
for non-enveloped
viruses
Subcuvia® (Baxalta, Shire Cold ethanol S/D Liquid ≥90 16 NR NR ≤4.8
plc, Jersey)b Glycine
Polyclonal IgG concentrate for large volume application at a single site
HyQvia® (IGHy) (Baxalta, Cold ethanol Liquid
Shire plc, Jersey) HyQvia is a two- The IgG component of HyQvia corresponds to Gammagard liquid (for details see Table 13.2)
component preparation HyQvia is applied sequentially, i.e., first the hyaluronidase than the IgG concentrate
with IgG concentrate
(10%) and recombinant
human hyaluronidase
(rHUPH20), 160 U/mL
Polyclonal IgG concentrates registered for i.m. and s.c. use
Gammaplex® 5% and 10% For details regarding Gammaplex see Tables 13.2 and 13.3
Polyclonal IgG concentrates intended for i.v. use applied s.c.c
Carimune® NF For details on Carimune NF, see Table 13.3
Gammagard® liquid For details regarding Gammagard liquid, see Table 13.2
without hyaluronidase
Gamunex® For details regarding Gamunex, see Table 13.1
Polyclonal IgG concentrates intended for i.m. use applied s.c.
BayGam® BayGam is also marketed as GammaSTAN; see below
(continued)
187
188

GammaQuin® Cold ethanol NR Liquid for i.m. or s.c. ≥90 16 None NR ≤6
Glycine
GamaSTAN® S/D (Grifols, Cold ethanol Precipitation, Liquid NR 15–18 None 6.4–7.2 NR
Grifols SA, Spain)) filtration ultrafiltration Glycine0.21–0.32 M
Diafiltration
SCIG is well suited individualizing immunoglobulin dose to enhance outcomes (Shapiro et al. 2017)
BPL Bio Products Laboratory, CSL Commonwealth Serum Laboratories, S/D solvent/detergent treatment in order to inactivate enveloped viruses
a
For details about polishing steps, please refer to Chap. 12
b
Shire in German recommends to replace Subcuvia® in favor of Cuvitru® or HyQvia®. It is foreseeable that in the future Subcuvia® will be taken from the mar-
ket, as it happened with Vivaglobin® in favor of Hizentra®
c
In the 1990s IVIGs were applied in order to prevent the use of those days mercury containing 16% i.m. preparations
P.J. Späth
Table 13.5 Some human hyperimmune globulin preparations
Strength Virus inactivation
of the IgG Ascertained and removal
in the specific steps part of the Stabilizer
solution IgG antibody manufacturing sugar Sodium
Specificity Brand name/manufacturer (mg/mL) Administration form content content process content content pH IgA content
Anti-toxin/toxoid polyclonal hyperimmune globulins
Tetanus Tetanus immunoglobulin-VF (CSL 55–65 Liquid for i.v. ≥98 4000 IU/ Low pH Maltose NR 4.25 <0.5 mg/
toxin Behring, CSL Ltd., Australia) vial Virus filtration 292 mmol/L mL
http://www.cslbehring.com.au/
docs/436/447/Tetanus%20IVIG-VF%20
AU%20PI%2014.00.pdf
Anti-viral polyclonal hyperimmune globulins
Human Cytotect® CP (Biotest, Biotest Pharma 50 Liquid for i.v. ≥96 100 PEI U./ Yes Glycine NR NR ≤2 mg/mL
CMV GmbH, Germany) mL May be of None
http://www.biotest.de/en/data/pdf/ limited value
therapie/produkte/spc-cytotect_cp_ against
en-nov2012.pdf non-enveloped
viruses
Cytogam® (CSL Behring, CSL Ltd., 5 Liquid for i.v. NR NR S/D Sucrose 5% 20– Trace
Australia) Human 30 mEq/L amount
https://www.fda.gov/downloads/ albumin 1%
BiologicsBloodVaccines/
BloodBloodProducts/ApprovedProducts/
LicensedProductsBLAs/
FractionatedPlasmaProducts/
UCM197962.pdf
http://labeling.cslbehring.com/PI/US/
Cytogam/EN/Cytogam-Prescribing-
Information.pdf
CMV immunoglobulin-VF (CSL 55–65 Liquid, for i.v. 1.5 million Yes 292 mmol/L 4.25 <0.5 mg/
Behring, CSL Ltd., Australia) units per maltose mL
http://www.cslbehring.com.au/docs/359/8/ vial
CMV%20Immunoglobulin%20AU%20
PI%2016.00.pdf
189
(continued)
190
Strength Virus inactivation

of the IgG Ascertained and removal
in the specific steps part of the Stabilizer
solution IgG antibody manufacturing sugar Sodium
Specificity Brand name/manufacturer (mg/mL) Administration form content content process content content pH IgA content
Hepatitis Hepatect® CP (Biotest, Biotest Pharma 50 Liquid for i.v. ≥96 50 I.U./mL Yes Glycine NR NR 2 mg/mL
B surface GmbH, Germany) May be of limited None
antigen https://www.medicines.org.uk/emc/ value against
(HBsAg) medicine/23171 non-enveloped
viruses
Zutectra® (Biotest, Bitest Pharma 150 Prefilled 5 mL ≥96 500 I.U./ Yes Glycine NR NR 6 mg/mL
GmbH, Germany) syringes for s.c. mL May be of None
http://www.ema.europa.eu/docs/en_GB/ limited value
document_library/EPAR_-_Product_ against
Information/human/001089/ non-enveloped
WC500073708.pdf viruses
Hepatitis immunoglobulin-VF (CSL 160 Liquid for i.m. ≥98 ≥100 IU/ Yes 22.5 mg/mL 6.6 NR
Behring, CSL Ltd., Australia) mL glycine
http://www.cslbehring.com.au/
docs/413/735/Hepatitis%20B%20
IMIG-VF%20AU%20PI%208.00.pdf
Human Varitect® CP (Biotest, Biotest Pharma 50 Liquid for i.v. ≥96 25 IU/mL Yes Glycine 2 mg/mL
varicella GmbH, Germany) May be of limited
zoster http://www.choroby-zakazne.pl/uploads/ value against
Varitect%20CP%20ang.pdf non-enveloped
viruses
Zoster immunoglobulin-VF(CSL 160 Liquid, ≥98 200 IU/vial Pasteurisation Glycine 6.6 NR
Behring, CSL Ltd., Australia) i.m. Virus filtration 22.5 mg/mL
http://www.cslbehring.com.au/ s.c.
docs/118/175/Zoster%20IMIG-VF%20
AU%20PI%209.00.pdf
Varzig® (Aptevo BioTherapeutics, NR Liquid for i.m. use, NR 125 IU/vial S/D Maltose 5.0– <0.04 mg/
Emergent BioSolutions Inc., USA) prepared by Virus filtration 10% 6.5 mL
http://varizig.com/VARIZIG%20 anion-exchange Polysorbate
P.J. Späth
LQ%20USPI-Aptevo.pdf chromatography 80 0.03%

Anti-blood group polyclonal hyperimmune globulins
Rhesus D Rhophylac® (CSL Behring CSL Ltd., 20 Liquid for i.v. or ≥95% 1500 IU per S/D Human ≤0.25 M NR <0.005 mg/
antigen Australia) i.m. vial Chromatography albumin mL
of red http://labeling.cslbehring.com/PI/US/ Pre-filled syringes Virus filtration 10 mg/mL
blood Rhophylac/EN/Rhophylac-Prescribing- A Glycine
cells Information.pdf chromatographically 20 mg/mL
isolated product
Rh(D) immunoglobulin-VF (CSL 10 or 30 Liquid for i.m. ≥98 250 or Pasteurization Glycine 6.6 NR
Behring, CSL Ltd., Australia) 625 IU per Virus filtration 22.5 mg/mL
http://www.cslbehring.com.au/ vial
docs/713/52/Rh(D)%20
Immunoglobulin-VF%20AU%20PI%20
12.00.pdf
WinRho® SDF (Aptevo NR Lyophilized powder NR 600, 1500 S/D Glycine 0.04 M NR 0.005 mg/
BioTherapeutics, Emergent for i.v. after or 5000 IU/ Virus filtration 0.1 M mL
BioSolutions Inc., USA) reconstitution vial Maltose
http://www.winrho.com/pdfs/ A product primarily 10%
WinRho_SDF_PI_Aptevo.pdf manufactured by Polysorbate
chromatographic 80 0.03%
method
The list is far from being complete

i.m. intramuscular, i.v. intravenous, S/D solvent/detergent treatment
191
192 P.J. Späth
Table 13.6 “Domestic” IgG concentrates. Toll-manufactured products might be widely used in
some of the countries listed
IgG concentrate (manufacturer, financial Main fractionation method/
background) reference
Australia and New Normal immunoglobulin-VF (CSL Formulation: Liquid for i.m.
Zealand Behring, CSL ltd., Australia) IgG content: ≥98
All “local” Strength of the solution for
products toll infusion: 160 mg/mL
manufactured Stabilizer: Glycine 22.5 mg/
mL
pH: 6.6
Various “Australian & New Zealand (Young et al. 2017)
Immunoglobulins” (CSL Behring
(Australia) Pty ltd)
China Human immunoglobulin (IM) (Shanghai
Raas, blood products Co. Ltd., China)
Gammaraas (Shanghai Raas, Blood Formulation: Liquid for i.v. at
Products Co. Ltd., China) pH of 4 with sorbitol
Strength of the solution for
infusion: 50 mg/mL
IgG content: ≥95
Pathogen safety: Low pH and
virus filtration
Shelf life: 36 mo at 2–8 °C
This product has a market
penetration in some countries
beside China
Human hepatitis B immunoglobulin Ascertained specific IgG
(IM) (Shanghai Raas, Blood Products content: 100, 200, 400 IU per
Co. Ltd., China) flask
Human tetanus immunoglobulin (IM) Ascertained specific IgG
(Shanghai Raas, Blood Products Co. content: 250 IU/vial
Ltd., China)
Human rabies immunoglobulin (IM) Ascertained specific IgG
(Shanghai Raas, Blood Products Co. content: 100 IU/mL
Ltd., China)
Normal human immunoglobulins Application: i.v.
A comparative study on
concentrations of antibodies
against β-amyloid 40/42
monomer and oligomers in 11
Chinese IVIGs
(Ye et al. 2017)
Cuba Intacglobin (Macías-Abraham et al. 2016)
13 Current IgG Products and Future Perspectives 193
India Immunorel (Reliance Life Science,
India)
Globucel (INTAS Pharma, Ahmedabad, Formulation: Liquid
India) Strength of the solution for
infusion: 50 mg/mL
Seroglob (Virchow/Gsk Healthcare Pvt. Strength of the solution for
Ltd.) infusion: 50 mg/mL
Immuglo (Hemarus Therapeutics Strength of the solution for
Limited, Hyderabad, India) infusion: 50 mg/mL
V-immune (Virchow Healthcare Pvt. Formulation: Liquid
Ltd. Mumbai, India) Strength of the solution for
infusion: 50 mg/mL
Japan Kenketsu Globulin “KAKETSUKEN” Formulation: Freeze-dried,
(Nihon Pharmaceutical Co., Takeda enzymatically degraded to the
Pharmaceutical Company Ltd.) F(ab)2 part by harsh pepsin
digestion
Kenketsu Venilon-I (Nihon Formulation: Freeze-dried,
Pharmaceutical Co., Takeda chemically modified by
Pharmaceutical company Ltd.) S-sulfonation
Glovenin-I (Nihon Pharmaceutical Co., Formulation: Freeze-dried,
Takeda pharmaceutical company Ltd.) polyethylene glycol-treated
human normal
immunoglobulin
DRIED HB GLOBULIN for Formulation: Freeze-dried
l.M. Injection 200 units INICHIYAKU Strength of the solution for
infusion: 200 mg/mL
Ascertained specific IgG
content: 250 U/vial
TETANUS GLOBULIN for Ascertained specific IgG
l.M. Injection 250 units INICHIYAKUJ content: 250 U/vial
ANTl-D GLOBULIN for l.M. Injection Ascertained specific IgG
1000 INICHIYAKUJ content: 1000 U/vial
South Africa Intragama (National Bioproducts, South (Peter et al. 2014)
Africa)
Polygamb (National Bioproducts, South (Peter et al. 2014)
Africa)
(continued)
194 P.J. Späth
South Korea IV-globulin SN™, (Green Cross Fractionation: Cold ethanol
Corporation, Yongin, Korea) and DEAE-sepharose
chromatography
Formulation: Liquid, maltose
at 100 mg/mL
Pathogen safety: S/D
treatment and virus filtration
Strength of the solution for
infusion: 50 mg/mL
Marketed in Korea, Brazil,
India, and Iran (Yoon et al.
2017; Stein et al. 2015)
Gamma globulin an i.m. concentrate (Tejada-Strop et al. 2017)
GC5101B (Green Cross Corporation, A Green Cross product
Yongin, Korea, and the Sungkyunkwan purified further (Park et al.
University, Suwon, Korea) 2017)
Furthermore, in several of the countries “nondomestic” products listed in the above tables are
available. The table is not a complete listing of all domestic products
BPL Bio Products Laboratory, CSL Commonwealth Serum Laboratories, LFB Laboratoire de
Fractionnement Bilogique, http://www.cslbehring.com.au/products/product-finder.htm
a
In Australia and New Zealand, the predecessor preparation of Intragam P was Intragam
b
Under the trade name Polygam the American Red Cross sold Gammagard in the USA; after the
transmission of HCV, the product is no more on the market
immunomodulatory potential of IVIGs was first reported (Imbach et al. 1981).
Following a distribution agreement with Sandoz, the product was renamed
Sandoglobulin. Sandoglobulin was the first IVIG which came onto the US market
after having been tested in the first IVIG registration study ever performed
(Cunningham-Rundles et al. 1984). Sandoglobulin was also the first product that
underwent large-scale virus filtration during manufacturing (Kempf and
Morgenthaler 1999). Sandoglobulin®/Sandoglobulin® NF is identical to
Carimune®/Carimune® NF and is also identical to Panglobulin®/Panglobulin®
NF. Panglobulin®/Panglobulin® NF was toll manufactured for the American Red
Cross (ARC). Panglobulin® /Panglobulin® NF is no more available. In contrast,
Sandoglobulin® NF/Carimune® NF is, at least in some markets, still available.
13.3 Future Perspectives
13.3.1 Polyclonal Immunoglobulin Preparations Different

from Presently Available Products
Fractionation of plasma to stable blood products generates a series of side fractions

some of which contain IgA and IgM. An investigational preparation for topical use
enriched in IgA (IgABulin) was clinically tested in a few patients; however,
following a company merger and the outcome of a Cochrane systematic review of

the project was not further pursued (Foster and Cole 2004). Polyclonal IgA isolated
from plasma contains a few percentage of a polymeric (dimeric) form of IgA (pIgA).
The combining of pIgA or of plasma-derived polyclonal IgM with recombinant
secretory component to form secretory-like IgA or IgM was reported recently
(Longet et al. 2013; Longet et al. 2014). To the best of my knowledge not trials in
man followed. A recent review regarding the clinical effects in man of human-
derived IgM came to the conclusion that the hard facts in knowledge are marginal
despite a bunch of clinical data obtained with a chemically modified concentrate
enriched in IgM and IgA (Späth et al. 2017). Nevertheless, some progress is made.
Biotest AG (Germany) developed a follow-up product to the existing chemically
modified IgM/A-enriched product, termed “BT086.” Clinical studies addressing
community- acquired pneumonia with BT086 have been completed (EudraCT
Number: 2010-022380-35 (https://www.clinicaltrialsregister.eu/ctr-search/
search?query=BT086) and Clinical Trail Identifier NCT01420744 (https://clinical-
trials.gov/ct2/show/NCT01420744?term=CIGMA&rank=1; both accessed June
2017). The results of this study are eagerly awaited. As BT086 has an isotype distri-
bution of approximately 54% IgG, 23% IgA, and 23% IgG (mean values), enrich-
ment in products of IgM above 25% is an unmet interest. A more recent patent
addresses a possible way how to obtain such highly enriched IgM (Rentsch 2000);
however no follow-up publications were found.
13.3.2 Resistance to Antibiotics and Polyclonal IgG Therapy
Outside the field of primary immune deficiencies, the clinical use of IgG concen-
trates in bacterial infection is almost inexistent. Table 13.5 does not list a single
antibacterial hyperimmune globulin. However, this might change. There are aston-
ishingly few reports which preemptively address a possible last resort role of IgG
therapy in fighting antibiotic-resistant/multiresistant infections (Diep et al. 2016;
Farag et al. 2013). Interest in IgG suddenly might awake, i.e., as soon as the spread-
ing of the mrc-1 gene located on plasmids of Gram-negative bacteria accelerates.
The mrc-1 gene has broken away the very last line of defense against antibiotic-
resistant Gram-negative bacteria (Liu et al. 2016).
13.3.3 Polyclonal Immunoglobulin Preparations for Infectious

Complications in Emerging Therapies
The past decade has seen a rapid appearance of innovative new immunosuppressive
and immunomodulatory drugs in clinics with currently three broad classes of bio-
logic therapies, i.e., tumor necrosis factor-alpha inhibitors, lymphocyte modulators,
and interleukin inhibitors. Therapeutic monoclonal antibodies, peptides, and fusion
proteins or engineered chimeric antigen receptor (CAR)-T cells are the basis of these
new therapies (Batlevi et al. 2016). Combination of these with well-established
196 P.J. Späth
therapies for, e.g., hematological malignancies, increases efficacy however also is

associated with severe infectious complications. The introduction of these modern
drugs is continuously generating an increasing number of clinical conditions that are
associated with an increased risk of severe/opportunistic/recurrent infections, reacti-
vation of latent viruses, and appearance of secondary autoimmune phenomena. The
resulting immunological deficiencies may involve innate immunity, adaptive T- and
B-cell immunity, or combinations of the three. The patients at risk thus are a hetero-
geneous population and include, beside HIV and hematological malignancies, malig-
nant solid tumors, hematopoietic stem cell transplant (HSCT) recipients, solid organ
transplant (SOT) recipients, patients on immunosuppressive or immunomodulatory
treatment for systemic autoimmune and inflammatory diseases, and other minor con-
ditions. When considering a single clinical condition treated with a single modern
therapeutic measure, the number of patients is small who might develop (severe)
infectious complications. However, the ever-increasing type of conditions treated
with the ever-increasing number of new drugs results in an ever-increasing number
of patients at risk for severe infectious complications. Particularly two subgroups of
patients with secondary susceptibility to severe infections might profit from therapies
with human immunoglobulin concentrates: (a) patients with secondary hypogam-
maglobulinemias showing an impaired vaccination response and (b) patients devel-
oping autoimmune conditions while on therapy for B-cell malignancies, prevention
of GvHD, or multiple sclerosis (e.g., following alemtuzumab treatment). Low level
of circulating IgG is not an indication for therapy in such conditions. In contrast,
recurrent/opportunistic infections and a proven failure of specific antibody produc-
tion after vaccination are indications for replacement therapy.
In summary, the evolution of new clinical conditions complicated by susceptibil-
ity to severe/recurrent/opportunistic infections is rapid, and the number of patients
at risk is steadily increasing. IgG therapies might have clinical role in those thera-
pies which directly or indirectly target the B-cell lineage and plasma cells.
13.3.4 The Combination for Improved Clinical Success

of Polyclonal IgG Concentrates with Modern Drugs
Targeting Immune Cells
There is an increased body of literature that reports the successful combination of

polyclonal IgGs with modern drugs that target immune cells. These combinations
should not be confounded with combinations where the immunosuppressive effect of
a modern therapy has to be corrected by passive immunization (see above). The most
reports relate to IVIG in combination with rituximab, bortezomib, or tocilizumab
either for desensitization prior to solid organ transplantation (SOT) or posttransplant
immunosuppression in highly sensitized patients (Jeong et al. 2016; Jordan et al.
2016; Perez et al. 2017; Ruangkanchanasetr et al. 2014; Vo et al. 2015). IVIG in
combination with rituximab has further been used in peripheral neuropathies (Oktem
et al. 2016) and in a few other conditions. Usually only severe, refractory cases are
receiving combination therapies, and hence the number of treated patients is small.
13.3.5 On the Edge Between Polyclonal and Recombinant

Products
The “fragment crystallizable” (Fc) together with the hinge region of immunoglobu-
lin molecules mediates the effector functions of the molecules. For IgG the
Fc-fragment accomplishes the binding to the Fcγ receptors and the binding of the
complement component C1q (initiation of the classical pathway of complement).
The hinge region is the site where complement components C3 and C4 are bound.
Already in the first report on immunomodulatory potential of polyclonal immuno-
globulin preparations, an important role for the Fc-fragments was recognized (cited
from (Imbach et al. 1981): “In one patient with acute ITP 0.5 g of a pepsin-treated
gammaglobulin [F(ab')2]/kg body-weight/day on 3 consecutive days did not influ-
ence platelet count, whilst a single dose of 0.4 g of Ig-SRK/kg body-weight raised
counts from 1.7 to 5 × 109/L within 6 h and to 12 × 109/L within 18 h.” Interestingly
enough, there exists one single study using Fc-fragment of IgG to treat ITP (Debré
et al. 1993). The study was successful, was never repeated, and, nevertheless,
together with other in vitro or in vivo studies, kept alive the interest about the rele-
vance in immunomodulation by Fc- fragments, the F(ab)- fragments, or the intact
IgG molecules. In a series of very elegant studies a research group around Jeffrey
Ravetch, The Rockefeller University, New York, from 2001 onward (Samuelsson
et al. 2001) added and continuously refined their concept how Fc-fragments might
take a role in immunomodulation: terminal sialic acid-guided interaction with
inhibitory Fcγ receptors being the key event (Kaneko et al. 2006; Schwab and
Nimmerjahn 2013; Schwab et al. 2015). A recent publication describes a robust,
controlled sialylation process to generate tetra-Fc–sialylated IVIG and its tenfold
enhanced anti-inflammatory effect in a mouse model of collagen antibody-induced
arthritis using a prophylactic dose (Washburn et al. 2015). The above concept is
based on mice experiments, and results are discussed controversially due to con-
flicting findings from other groups (Bazin et al. 2006; Campbell et al. 2014; Crispin
et al. 2013; Guhr et al. 2011; Käsermann et al. 2012; Leontyev et al. 2012; Leontyev
et al. 2012; Othy et al. 2014; von Gunten et al. 2014; Yu et al. 2013; Schneider et al.
2017). In humans an exclusive role of terminal sialic acid-guided interaction with
inhibitory Fcγ receptors as anti-inflammatory/immunomodulatory principle remains
open. The mechanism of action of IgG concentrates might involve such interaction
however; it is likely that this interaction is not sufficient to explain the anti-
inflammatory/immunomodulatory potential of polyclonal normal IgG concentrates.
Nevertheless, in humans terminal sialylation of Fc-fragments might be involved in
some immunoregulatory processes. As an example, in pregnancy partial suppres-
sion of the maternal immune system is required to ensure tolerance of the fetus
(Trowsdale and Betz 2006). In successful pregnancies, an increase in IgG Fc-linked
198 P.J. Späth
N-glycan galactosylation and sialylation and decrease after delivery were observed
(Jansen et al. 2016). How far this and no other changes in glycans during pregnancy
is responsible for tolerance remains open.
The interest in the biological role of Fc-fragments of IgG and their
N-glycosylation is unbowed. The aim is to reproduce effector mechanism of poly-
clonal IgG at lower doses, and this has opened out into generation of engineered
forms of Fc-fragments and their N-glycans. Multimerized recombinant Fc is one of
the options. Momenta Pharmaceuticals is, among others, working on a trimeric Fc
structure, and the start of a phase I study is planned for 2017 (Ortiz et al. 2016).
Another avenue is pursued by Gliknik. GL-2045 is a stradomer, a multimeric IgG2
hinge-Fc in preclinical safety studies. The hinge region allows the binding of com-
plement C3 and C4 and is an inhibitor of complement-mediated cytotoxicity (Zhou
et al. 2017). HexaGard™ is a hexameric IgG1Fc with IgM tailpiece [L309C/
H310L], i.e., is a biomimetic substitute of IVIG for triggering inhibitory receptors
involved in controlling unwanted inflammation in autoimmune disease (http://
www.lstmed.ac.uk/news-events/events/hexagard-a-biomimetic-of-intravenous-
immunoglobulin-ivig-for-the-treatment-of; accessed July 2017). UCB Celltech is
working on a hexameric, hybrid IgG1/4Fc IgM tailpiece. A subject-blind, investi-
gator-blind, randomized, placebo-controlled, first-in-human study evaluates the
safety/tolerability and pharmacokinetics of single ascending intravenous and sub-
cutaneous doses of UCB7858 in healthy subjects (https://clinicaltrials.gov/ct2/
show/NCT02879877; accessed July 2017). Argenex has created a monomeric
IgG1Fc with high affinity for FcRn (ARGX-113). ARGX-113 is in phase II trials
in myasthenia gravis and ITP. ARGX-113 enhances degradation of circulating
disease-causing autoimmune antibodies by competing for binding to the Fc recep-
tor of the newborn (FcRn), an MHC class I-like Fc receptor (Yu and Lennon 1999).
FcRn is the receptor on endothelial cell that recirculates IgG and prolongs the half-
lives in circulation to above 2 days (except IgG3) (Andersen and Sandlie 2009).
FcRn is also the receptor for transplacental passage of IgG from mother to fetus
(Firan et al. 2001). A more broad review regarding these new developments was
published recently (Zürcher et al. 2016). More recently engineered IgG Fc domains
that bind C1q but not effector Fcγ receptors mediated the clearance of target cells
with kinetics and efficacy comparable to those of the FcγR-dependent effector
functions, while they circumvented certain adverse reactions associated with FcγR
engagement (Lee et al. 2017).
Beside the avenue of anti-inflammation/immunomodulation, even host defense
was addressed by engineered Fc-fractions. Lysibodies are IgG Fc fusions with
lysin-binding domains targeting Staphylococcus aureus cell wall carbohydrates for
effective phagocytosis (Raz et al. 2017). Lysibodies target bacterial structures con-
served over the evolution and thus escape mutations have a lesser chance for
success.
In summary, innovative clinical trials with polyclonal normal IgG are not shin-
ing on the horizon. A considerable effort is taken to elucidate the biologic role of
the Fc-fraction of IgG. For this purpose various, often multimeric, Fc-fragments

with altered glycosylation are engineered. Some of these have entered phase II
studies.
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Part III
Immune Thrombocytopenia: The First
Immunomodulatory IgG Treatment
Updates in Immune Thrombocytopenia:
Terminology, Immunomodulation 14
and Platelet Stimulation, and Clinical
Guidelines and Management
Cindy Neunert
14.1 Introduction
ITP results from antibody-mediated platelet destruction as well as decreased pro-

duction. Recently our understanding of the pathophysiology of ITP has expanded,
increasing therapeutic options. This section will outline new terminology, describe
the pathophysiology, and highlight treatment options and guidelines for
management.
14.2 ITP Terminology
In 2008 an International Working Group (IWG) consisting of ITP experts provided

consensus regarding the terminology to be used in reference to patients with ITP
(Rodeghiero et al. 2009). While the primary purpose of this document was to estab-
lish uniform research outcomes, much of the terminology applies to clinical care.
The highlights of this are outlined below:
• Diagnosis: Now based on a platelet count of <100 × 109/L, given that healthy

individuals may have platelet counts between 100 and 150 × 109/L with low like-
lihood of progression (Rodeghiero et al. 2009)
• Disease Duration:
–– Newly diagnosed—diagnosis until 3 months
–– Persistent—between 3 and 12 months
–– Chronic—greater than 12 months
C. Neunert, MD, MSCS

Columbia University Medical Center, 3959 Broadway, CHN 10-03, New York, NY 10032,
USA

206 C. Neunert
• Severe ITP: Patients who have clinically relevant bleeding defined as the pres-
ence of bleeding symptoms either at presentation or subsequent new bleeding
symptoms which require therapeutic intervention with a different platelet-
enhancing agent or an increased dose of current medication. Patients can present
with emergency bleeding events. These include life-threatening bleeding such as
intracranial hemorrhage, abdominal bleeding, and/or bleeding in the setting of
trauma. Physicians should consider symptoms and history in order to promptly
recognize and manage these events.
• Refractory ITP: Patients who have failed splenectomy or relapsed following
splenectomy and have severe ITP or have a risk of bleeding that in the opinion of
the physician requires therapy
• Response Criteria:
–– Complete response—any platelet count 100 × 109/L AND resolution of
bleeding
–– Response—any platelet count 30 < 100 × 109/L AND at least doubling of the
baseline count and resolution of bleeding
–– No response—any platelet count <30 × 109/L OR less than doubling of the
baseline count and resolution of bleeding
• Limitations to Definitions:
–– No established bleeding score to determine exact definition of severe disease
based on bleeding symptoms alone (Neunert and Arnold 2015)
–– Definition of refractory requiring that patients have undergone and failed
splenectomy may not apply to certain populations such as children or those
with a contraindication to splenectomy
–– The need to account for the variable time to response for different treatments
–– The need to account for therapies which require ongoing treatment (such as
TPO-RA) compared to those that are given at a single time point (such as
IVIg)
14.3 Immunomodulation and Platelet Stimulation
14.3.1 Pathophysiology of ITP
The earliest understanding of ITP came from the experiments of Dr. Harrington. In
1950 Dr. Harrington injected himself with blood from a patient with ITP (Harrington
et al. 1951). He had a rapid decline in his platelet count, giving some of the first
evidence that ITP was a blood disorder. Later it was understood that this “blood fac-
tor” was antibodies interacting with the surface of the platelets and causing them to
be removed from circulation by Fc gamma receptors on splenic macrophages (van
Leeuwen et al. 1982). More recently, however, we have also come to appreciate the
highly complex process that appears to be disrupted in patients with ITP. These take
on two distinct pathways: reduced tolerance to self and increased immune activation
(Panitsas et al. 2004; McKenzie et al. 2013; Olsson et al. 2003).
14 Updates in Immune Thrombocytopenia 207
• Reduced Tolerance to Self

–– Reduced T regulatory cells
–– Reduced B cell inhibitory Fc receptors
• Increased Immune Activation
–– Increased Th1/Th2 ratio
–– Increased Th17 cells
–– Direct increased in cytotoxic T cells against platelets
14.3.2 Immunomodulation
These discoveries have increased our therapeutic options with immunomodulatory

medications. Table 14.1 outlines therapies available for ITP, their predominant
mechanism of action, and available clinical data (Cuker and Neunert 2016).
Table 14.1 Immunomodulatorytreatment for ITP

Agent Mechanism of action Dosing Response data
First-line immunotherapy
Corticosteroids Upregulation of Prednisone: 1 mg/kg/
anti-inflammatory day for 21 days
proteins followed by a taper
Downregulation of Dexamethasone:
inflammatory proteins 40 mg/day for
4 days × 2–4 weekly
cycles
Intravenous Fc receptor blockage 0.8–1.0 g/kg × 1
Immunoglobulin (IVIg) Synergistic effects of
innate and adaptive
immunity
Anti-D immunoglobulin Rh red cell sensitization 50–75 mcg/kg × 1
causing Fc receptor
competition
Second-line immunotherapy
Splenectomy Removal of site of N/A 60–80%
platelet destruction
Rituximab Anti-CD20 monoclonal 375 mg/m2/dose 60% overall
antibody weekly × 4 doses 40% complete
Third-line immunotherapy
6-Mercaptopurine Inhibits purine 50–75 mg/m2 Qday 83%
nucleotide synthesis
Azathioprine Inhibits purine 1–2 mg/kg Qday (max 40–60%
nucleotide synthesis 150 mg/day)
Cyclosporin A Inhibits calcineurin 2.5–3 mg/kg BID 30–60%
blocking transcription (titrate to level of
of cytokine genes 100–200 ng/mL)
(continued)
208 C. Neunert
Agent Mechanism of action Dosing Response data
Cyclophosphamide Cross-links DNA 0.3–1.0 g/m2/dose 24–85%
causing inhibition of every 2–4 weeks × 1–3
DNA replication doses and then 50–200
Mechanism in ITP orally after response
unclear can tapered to 50 mg
daily
Vincristine Binds to tubulin and Vincristine: 1–2 mg IV 10–75%
inhibits cell division weekly × 3–6 doses
Vinblastine Vinblastine: 10 mg IV
weekly × 3 weeks
Dapsone Blockade 75–100 mg Qday 40–75%
myeloperoxidase
Mechanism unclear in
ITP
Mycophenolate mofetil Inhibits inosine-5′- 250–1000 mg BID 11–80%
monophosphate
dehydrogenase and
impairs T and B
lymphocyte
proliferation
Danazol Unclear in ITP 50–800 mg/d orally 10–70%
divided into 2–4 doses
per day
14.3.3 Platelet Stimulation
It has more recently been shown that IgG from patients with ITP binds to and sup-
presses megakaryocyte production (Chang et al. 2003; McMillan et al. 2004).
Megakaryocytes in the bone marrow also demonstrate abnormal changes consistent
with apoptosis and inflammation (Houwerzijl et al. 2004). These discoveries lead to
the development of thrombopoietin receptor agonists (TPO-RAs), which stimulate
platelet production.
There are two current TPO-RAs that have been widely studied in adult and pedi-
atric trials, eltrombopag and romiplostim, each outlined below.
• Eltrombopag
–– 25–75 mg PO Qday (titrated based on platelet count)
–– Results of mean platelet counts during clinical trial (Saleh et al. 2013)
N=253b N=218b N=147b N=32b
200
Median Platelet Count (×103/µL)
150
100
50
0
BL 1 2 3 4 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 156
Weeks
Number of patients 299290285272 272 243 166 128 107 94 68 77 78 71 72 61 50 43 31 12 12 15 12 11 10
• Romiplostim
–– 1–10 mcg/kg SC weekly (titrated based on platelet count)
–– Results of mean platelet counts during clinical trial (Kuter et al. 2013)
350
300
250
Platelet Count x 10 /L
Median (01-03) 9
200
150
100
50
0
0 6 15 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 184 192 200 206 216 224 232 240 248 256 264 272
Study Week
n = 291 257 242 233 227 228 210 210 194 156 129 110 100 95 92 86 83 81 82 80 75 74 67 57 45 41 31 26 22 23 19 17 13 14 11
• Major Adverse Events of the TPO-RAs

–– Thrombocytosis, bone marrow reticulin, and thrombosis can occur with both
medications.
–– Hepatic toxicity can occur with eltrombopag.
–– Rebound thrombocytopenia and bleeding can be seen with drug
discontinuation.
210 C. Neunert
14.4 Clinical Guidelines and Update on Management
Current clinical practice guidelines exist for the management of ITP (Neunert
et al. 2011; Provan et al. 2010). Most guidelines reference treatment for patients
with newly diagnosed ITP who require first-line therapy and then additional guid-
ance for those who develop more long-standing ITP and/or become refractory to
first-line therapy. General management for adult and pediatric patients is outlined
below:
• Newly Diagnosed ITP in Adults

–– Treatment should be considered for patients with a platelet count <30 × 109/L,
and treatment is rarely needed for patients with a platelet count >50 × 109/L.
–– Longer courses (≥21 days with a taper) of corticosteroids are the preferred
first-line treatment.
Physicians should be aware of the side effects associated with long-term cor-
ticosteroid use including but not limited to mood disorders, diabetes, hyper-
tension, weight gain, osteoporosis, and cataracts.
–– IVIg can be given along with corticosteroids if a more rapid increase in the
platelet count is required.
–– IVIg and anti-D immunoglobulin can be used if there is a contraindication to
corticosteroids.
• Newly Diagnosed ITP in Pediatrics
–– The majority of children with no or mild bleeding only do not require treat-
ment regardless of the platelet count.
–– If treatment is necessary, patients can be treated with IVIg, anti-D immuno-
globulin, or corticosteroids.
–– IVIg or anti-D immunoglobulin is preferred if a more rapid increase in the
platelet count is required.
• Persistent, Chronic, and Refractory ITP in Adults
–– Further treatment is not necessary for asymptomatic patients with a platelet
count >30 × 109/L unless there is concern about additional risk factors for
bleeding or impaired health-related quality of life.
–– Splenectomy should be considered for all adults who have failed corticoste-
roids and/or additional treatment regimens.
–– TPO-RAs can be considered in patients who fail splenectomy, have a contraindi-
cation to splenectomy, and/or have failed at least one additional therapy.
–– Rituximab can be considered for patients who have failed at least one addi-
tional therapy.
• Persistent, Chronic, and Refractory ITP in Pediatrics
–– Rituximab may be considered as an alternative to splenectomy in children and
for those who fail splenectomy and/or who have significant ongoing bleeding
despite treatment with at least one additional therapy.
–– High-dose dexamethasone may be considered as an alternative to splenec-
tomy in children and adolescents and for those who fail splenectomy and/or
who have significant ongoing bleeding despite treatment with at least one
additional therapy.
–– Splenectomy can be considered for children and adolescents with chronic or

persistent ITP who have significant or persistent bleeding, lack of responsive-
ness to other therapies, and/or impaired quality of life.
–– Splenectomy and interventions with potentially serious complications should
be delayed for at least 12 months when feasible.
• Management Considerations
–– Anti-D immunoglobulin should not be given to patients with significant ane-
mia and requires a patient to be Rh+ in order to be effective. An average 2.0 g
decline in hemoglobin is seen following use. There have also been cases of
fatal intravascular hemolysis and disseminated intravascular coagulopathy
following use (Gaines 2005).
–– There is no evidence to support the specific platelet count threshold for treat-
ment in adult patients with ITP.
–– The ideal type, duration, and method of corticosteroid administration for
newly diagnosed adults with ITP are currently controversial with no differ-
ence in long-term outcomes between prednisone and dexamethasone
(Mithoowani et al. 2016).
–– Additional immunosuppressive agents (outlined in the section above) can be
considered for patients with refractory disease; however, the data on these
agents is minimal.
–– More recent evidence, not available at the time of guideline development,
supports the use of TPO-RAs in children with chronic disease (Grainger et al.
2015; Tarantino et al. 2016; Bussel et al. 2011, 2015).
–– The decision to treat and therapy selected should always involve consider-
ation of the patient preferences, bleeding symptoms, quality of life, and
comorbidities.
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Health-Related Quality of Life in Patients
with Immune Thrombocytopenia 15
Robert J. Klaassen and Nancy L. Young
15.1 Introduction
I walked into the examination room to see my second patient of the day—five other
families were quietly sitting in the waiting room awaiting their turn. She was a
15-year-old girl with immune thrombocytopenia (ITP) diagnosed just over 1 year
ago. Her platelet count ranged from 20 to 30 × 109/l, but she had not required any
emergency department visits or had any significant bleeding events over the past
6 months. All in all she seemed stable, so my plan was to continue with the current
treatment approach of “watch and wait”.
I was about to wrap up the visit when I noticed that she was looking dejected and
asked her how she was doing. She explained that she had been very active in rugby
before the diagnosis, and now she had no energy and spent most of her time on the
couch. I had, on multiple occasions, previously reassured her that she could fully
engage in all non-contact sports, but she still felt restricted and run down. Because
of this interaction, I changed my plan and discuss in detail the many second-line
treatment options available for chronic ITP.
This clinical example illustrates that patients’ perceptions contribute an essential
component of the history which in turn has a central bearing on the treatment plan.
It also shows that elucidating this information in the clinic setting often occurs by
chance. In this chapter, we will discuss options for a more intentional and system-
atic approach, using quality of life assessment tools.
R.J. Klaassen, MD (*)

Division of Hematology/Oncology, Department of Pediatrics, University of Ottawa,
Children’s Hospital of Eastern Ontario, 401 Smyth, Ottawa, ON, Canada, K1H 8L1
N.L. Young, PhD
School of Rural and Northern Health, Laurentian University, Sudbury, ON, Canada

214 R.J. Klaassen and N.L. Young
15.2 W
hat Is Quality of Life and Health-Related Quality
of Life (HRQoL)?
Quality of life (QOL) has been defined by the World Health Organisation as “an indi-
viduals’ perception of their position in life in the context of the culture and value sys-
tems in which they live and in relation to their goals, expectations, standards and
concerns” (WHOQOL Group 1993). They organised QOL into six domains: physical,
psychological, level of independence, social relationships, environmental and spiritual/
religion/personal beliefs. A more concise way of looking at QOL is the gap between our
expectations and our experience (Calman 1984). Further narrowing the concept is the
term health-related quality of life (HRQOL), which excludes some of the domains that
are generally not influenced by health services, such as spiritual beliefs and finances
(which may be relevant depending on the local health care system). In many instances,
the terms are used interchangeably, but we will focus this chapter on HRQOL.
15.3 Why Do We Need to Measure HRQOL?
Clinicians intuitively know that HRQOL is important in medical decision-making; this

is an essential component of the “art of medicine”. At the start of this chapter, I pro-
vided an example of a patient who “on paper” did not fulfil the criteria for going on to
second-line (potentially curative) therapy for ITP. It was only when I started delving
into her perception about her disease that I became aware of the impact of her illness
on her HRQOL. I have other patients with the exact same clinical phenotype, who were
perfectly content and did not recommend a change in course of their treatment.
The problem with this ad hoc approach to evaluating HRQOL is that important
deficits in HRQOL can easily be missed, especially when dealing with poorly com-
municative teenagers. A less perceptive clinician, or any one of us on a busy day,
may have walked out of the room without any further discussion. The alternative,
especially in the research setting, is to systematically collect data on HRQOL, typi-
cally using patient-reported outcome measures (PROs). This allows for a more stan-
dardised approach to looking at HRQOL and provides numeric comparisons over
time and between patients.
15.4 Types of Measures
Two general types of measures can be used to measure HRQOL: (1) generic and (2)
disease specific, each having their own advantages and disadvantages (Eiser and
Jenney 2007). Generic HRQOL tools can be used across disease groups and can
even be administered to “healthy” populations (Varni et al. 2003). This allows for
comparisons between different patient populations using the healthy population as
a reference. Because these tools can be used in many different settings, they are well
known to clinicians and are therefore easier to understand, with the resulting scores
being easy to interpret. Examples include the medical outcomes study short-form
15 Health-Related Quality of Life in Patients with Immune Thrombocytopenia 215
survey (SF 36) https://campaign.optum.com/optum-outcomes.html (Ware 2004),

which is commonly used in adults and the Pediatric Quality of Life Inventory
(PedsQL) http://www.pedsql.org/ in children (Varni et al. 2003).
Disease-specific HRQOL tools have the advantage of including specific ques-
tions related to the disease states that are not covered by generic tools, such as ITP-
related questions as in “I worried about my platelet count…” which is included in
the Kids ITP Tools (KIT) http://www.flintbox.com/public/project/3044 (Barnard
et al. 2003) but is obviously not present in the PedsQL. The main advantage of this
particular method of questioning is that a very detailed picture of the patients
HRQOL can be obtained, which is more sensitive to change over time (Klaassen
et al. 2007). The disadvantage of these tools is that comparisons between disease
groups are not possible, and since they are less well known, interpretation of the
scores may be less intuitive.
In general, both types of measures have unique value and should be included,
when possible. A generic tool will provide the broad perspective that allows com-
parison to other groups and the healthy population, whereas the disease-specific tool
allows a more detailed picture of the patient’s perception of their disease. The
respondent burden in a particular study or clinical setting must also be considered
and may require a choice be made to use one or the other approach.
The two available disease-specific measures routinely used in ITP include the
KIT for children (Klaassen et al. 2013) and the ITP-PAQ for adults (Mathias et al.
2007). Since these tools are crucial to the full understanding of HRQOL in this
disease, I will describe them in more detail.
15.4.1 The Kids’ ITP Tools (KIT)
The KIT was initially developed in North America specifically for children with ITP
and their parents (Barnard et al. 2003) and later refined (Klaassen et al. 2007; Klaassen
and Young 2010) and cross-culturally validated for other regions of the world
(Klaassen et al. 2013). It has three versions: one for the child (child self-report), where
the child is asked to focus on what he/she thought about and did; another for the parent
to complete on behalf of the child (parent proxy report), where the parent is asked to
focus on their child’s HRQOL; and finally one for the parent complete about them-
selves (parent impact report) to capture the HRQOL experience of the parent. All of
the versions consist of 26 questions with the total score ranging from 0 (worst) to 100
(best) HRQOL. There are no subscales. Data supporting the validity, reliability and
responsiveness of KIT has been published (Klaassen et al. 2007). The KIT has been
cross-culturally translated into 24 languages in 21 countries.
15.4.2 ITP-Patient-Administered Questionnaire (ITP-PAQ)
The ITP-PAQ is a questionnaire developed for use in adults with chronic ITP to
measure HRQOL, consisting of 44 items grouped into 10 scales: Physical Symptoms
(6 items), Bother-Physical Health (3), Fatigue/Sleep (4), Activity (2), Fear (5),
Psychological Health (5), Work (4), Social Activity (4), Women’s Reproductive
Health (6) and Overall Quality of Life (5) (Mathias et al. 2007, 2009). No summary
score is calculated. Each scale is scored from 0 to 100, with higher scores represent-
ing better HRQOL. It was found to be valid with moderate correlation to the SF-36
and was able to differentiate ITP patients on treatment from those off treatment
(McMillan et al. 2008). The ITP-PAQ can be used to describe the burden of illness
as well as an outcome measure to assess the efficacy or effectiveness of ITP
treatments.
15.5 Study Results
15.5.1 Paediatric
HRQOL has been assessed in a number of paediatric studies with intriguing

results—a review of 90 children with ITP found no correlation between the KIT,
the platelet count and bleeding scores, clearly indicating that quality of life is
perceived differently by the patient and family from the platelet count and
bleeding (Neunert et al. 2009). A further analysis of 217 children failed to find
a difference in self-reported KIT scores between children who received treat-
ment compared to those who were simply observed (Grainger et al. 2013). This
same study found that parents proxy-reported scores were significantly lower
for children with newly diagnosed ITP who were treated vs. not treated (mean
KIT scores of 48 and 64, respectively, p = 0.03) (Grainger et al. 2013). This
raises the possibility that treatment may be detrimental to patients’ HRQOL, or
conversely when parents perceive that their child’s HRQOL is low, they elect to
treat their child.
Of interest, multiple randomised trials of the thrombopoietin receptor agonist
(TPO RA) drugs have failed to show a significant improvement in HRQOL, based
on self-report or proxy report, when compared to placebo in a sample that demon-
strated significant improvements in the platelet count during the same period (Bussel
et al. 2015; Tarantino et al. 2016). The main reason for this finding was that there
was almost as much improvement in the placebo arm as the treatment arm, pointing
to the impact of participating in a clinical trial that lessens over the course of the
study. One pilot study of Romiplostim did show a significant improvement in paren-
tal burden in the treatment arm compared to placebo (change of +24 vs. −6,
p = 0.008) (Klaassen et al. 2011).
The one consistent finding across all studies is that patients with newly diag-
nosed ITP have lower HRQOL scores than patients with chronic ITP (Grainger
et al. 2013; Mokhtar et al. 2014) and that scores steadily increase with time after
initial diagnosis as many patients go into remission, and the patients and their fam-
ily adjust to the disease. Those patients who continue to have disease on follow up
understandably report lower HRQOL scores compared to those patients who go into
remission (Fig. 15.1) (Flores et al. 2017; Heitink-Polle et al. 2014).
a
100.0 *
*
80.0
Parental Disease Burden
60.0
40.0
20.0
*
0
Diagnosis 1 week 6 months 12 months
b Study Visit
* *
100.0
80.0
HRQoL Child
60.0
40.0
20.0
0
c Study Visit
* *
100.0
80.0
HRQoL Proxy
60.0 *
40.0
20.0
0
Study Visit
Fig. 15.1 Change in KIT scores for parent (a), child (b) and proxy (c) questionnaires from diag-
nosis through 12 months (parent, p = 0.009; child, p < 0.0005; proxy p = 0.001). Asterisks denote
statistically significant improvement in score from six pairwise comparisons (p < 0.008; 0.05/6).
Adapted from Flores et al. (2017)
15.5.2 Adult
Adults with ITP have consistently been shown to have lower HRQOL when com-
pared to the healthy population in studies measuring the SF36 in China, the USA,
the UK and Italy (Fig. 15.2) (Efficace et al. 2016; McMillan et al. 2008; Snyder
et al. 2008; Zhou et al. 2007) and the EQ-5D in the USA, the UK, France, the
Netherlands and Spain (Sanz et al. 2011; Snyder et al. 2008). In particular, between
five and seven of the eight domains of the SF36 were significantly lower, with only
bodily pain not affected (which makes sense clinically). General health, role physi-
cal (the ability to get things done), vitality (energy) and social functioning were the
domains consistently discrepant across studies from the control population, point-
ing to the impact of ITP on overall functioning (McMillan et al. 2008; Snyder et al.
2008; Zhou et al. 2007). When compared to other patient populations, ITP patients
had worse SF36 scores than patients with hypertension, arthritis and cancer, similar
scores to diabetes and higher scores than patients with congestive heart failure or
missing a limb.
As opposed to the situation in paediatrics, adult therapeutic studies have
clearly shown that second-line therapies result in significant improvements in
HRQOL when compared to placebo (George et al. 2009; Kuter et al. 2010; Sanz
et al. 2011). An analysis of two phase 3 randomised placebo-controlled European
studies of romiplostim that incorporated the EQ-5D, a health utility-based mea-
sure which can be used in economic evaluation, showed a significant
100
ITP patients Population norms
P < 0.001*
90
∆=.5.2
P < 0.001* P < 0.001*
80 ∆=-16.0** P = 0.347 ∆=-6.3 P = 0.002*
81.5 ∆=-1.7 ∆=-8.4* P = 0.417
76.1 75.2 P = 0.008 75.6 ∆=-1.1
70 P = 0.350 71.4
69.3 71.4 ∆=-4.0
∆=-3.0 69.1
60 62.7 63.9 65.2
61.4 59.7
59.0 57.2 56.5
50
40
30
20
10
0
Physical Role Bodily General Vitality Social Role Mental
Functioning Physical Pain Health Functioning Emotional Health
Fig. 15.2 Adjusted comparisons of SF-36 scales between pITP patients (overall population) and
general population norms. Legend Δ mean differences were adjusted for age, sex, education, geo-
graphic area and marital status: *The score difference exceeds the minimally important difference
(i.e. 8 points); **The score difference exceeds twice the minimally important difference; # statisti-
cally significant after Bonferroni adjustment (adjusted alpha = 0.05/8 = 0.00625). Adapted from
Efficace et al. (2016)
improvement over 6 months in the index score (0.05 vs. −0.03, p = 0.015) but did
not achieve significance with the visual analogue score (6.42 vs. 0.48, p = 0.066).
Using the disease-specific measure ITP-PAQ, an analysis of two American ran-
domised placebo-controlled trials, again of romiplostim, showed significant
improvement in seven of ten scales (Symptoms, Bother, Activity, Fear,
Psychological Health, Social Activity and Women’s Reproductive Health) when
compared to placebo (George et al. 2009). This was confirmed in an open-label
comparison of romiplostim to standard of care, which showed statistically sig-
nificant improvement in six scales (Symptoms, Bother, Activity, Fear,
Psychological Health, Social Activity) and Overall QOL for patients given
romiplostim. Eltrombopag has much less adult QOL data available, with the
landmark NEJM published in 2007 s howing no change in SF36 scores. This is
likely due to the fact that they did not incorporate a disease-specific tool into
their trials (Bussel et al. 2007).
15.6 Fatigue
A specific mention needs to be made of the issue of fatigue in ITP, since this is an
important symptom that has been reported in numerous studies in spite of the lack
of a clear pathogenic link to ITP (Efficace et al. 2016; Newton et al. 2011). A large
combined US and UK study published in 2011 of 653 adults using the Fatigue
Impact Scale (FIS) found significantly increased rates of fatigue when compared
to the healthy population (UK 39%, USA 22% with FIS score ≥40 vs. 2.5%,
respectively; p < 0.0001) (Newton et al. 2011). This was confirmed in an Italian
ITP study which used the Multidimensional Fatigue Inventory (MFI) and again
found significantly worse general, mental and physical fatigue as well as reduced
activity (p for all dimensions <0.001) (Efficace et al. 2016). Children experience
fatigue as well, with a study that administered the Fatigue Scale-Child, Adolescent
and Parent (FS-C, A, P) to 102 children with ITP showing that children as young
as seven experienced significantly more fatigue than the healthy population (Grace
et al. 2016).
ITP patients have been complaining about this symptom long before it was docu-
mented in the literature but was often dismissed by clinicians as unrelated to their
disease. A review article on the topic by Hill et al. proposed a pathogenic model
(Fig. 15.3) linking ITP with peripheral inflammation, bruising and activity restric-
tions, anaemia and iron deficiency, tied in with social factors and cognitive/behav-
ioural issues all contributing to fatigue (Hill and Newland 2015). Going forward,
assessment of fatigue needs to be included in the arsenal of patient-reported out-
comes for this illness.
Anaemia & iron Underlying disease Pre-morbid factors

deficiency (secondary ITP) and co- • Lack of activity
morbid medical disorders • Psychological symptoms
• Cytokine gene polymorphisms
ITP*
IFN-γ
B cell TNF-α
Tcell (Th1)
Peripheral CNS neuro-endocrine Fatigue

Inflammation disturbance
Platelets
MP
Social factors
• Work/carer
Drug therapies responsibilities
• Steroids • Social support
Sleep disturbance
• Immunosuppression
Cognitive/behavioural
• Illness beliefs
Bruising & activity • Perceived stress
restrictions • Mood
Fig. 15.3 Model of the pathogenesis of ITP-associated fatigue. ITP, immune thrombocytopenia;
B cell, autoantibody-producing B lymphocytes; T cell Th1, T lymphocytes with a T helper 1 polar-
isation; TNF-α, tumour necrosis factor alpha; IFN-γ, interferon gamma; MP, microparticles; CNS,
central nervous system. *T cells can attack platelets directly, secrete pro-inflammatory cytokines
and drive formation of autoreactive B cells. This results in antibody- and complement-mediated
platelet destruction. Platelet microparticles are prothrombotic and able to activate the pro-
inflammatory complement pathway. Adapted from Hill and Newland (2015)
15.7 Implementing HRQOL Tools in Clinical Practice
All of this leads to the recommendation proposed at the start of the chapter: How do
we implement a more intentional and systematic approach, using HRQOL assess-
ment tools? Unfortunately, most of the available HRQOL tools were developed for
the research setting and need to be further assessed in the clinic to determine if they
can be used for clinical decision-making. A number of investigators have been
focusing on this task, with the International Society of Quality of Life releasing a
user’s guide in 2015 for the implementation of patient-reported outcomes in clinical
practice http://www.isoqol.org/research-publications/isoqol-publications. Older lit-
erature has not clearly shown that using these tools in the clinic has influenced the
management of patients’ problems (Greenhalgh 2009), but fortunately that early
work has clarified different strategies to make this successful in the future. With the
widespread implementation of electronic health records by many clinicians, the
reality of integrating HRQOL tools into routine clinical practice has become tanta-
lisingly close.
Returning to my patient described in the introduction, after our discussion of
possible second-line ITP therapies, she elected to receive a course of rituximab.
Fortunately she experienced no significant side effects, and a few months later, she
was in remission with a respectable platelet count. Clinically she felt much better
and was smiling with renewed energy. A fortunate outcome that should hopefully
become more commonplace with the systematic integration of HRQOL tools into
the clinic.
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ITP in Childhood: Predictors of Disease
Duration 16
Carolyn M. Bennett
16.1 Introduction
Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized

by increased platelet destruction and decreased platelet production resulting in iso-
lated thrombocytopenia (Harrington et al. 1951; McMillan et al. 2004; Zufferey et al.
2017). ITP may present at any age, including infancy, but the natural history in chil-
dren usually differs from adults (Imbach et al. 2006; Kuhne et al. 2011). Most adults
with ITP have an insidious onset of disease without a preceding viral illness and
develop chronic disease that persists beyond 6–12 months, while the majority of chil-
dren will have self-limited thrombocytopenia that resolves completely within weeks
to months of presentation (Provan et al. 2010; Kuhne et al. 2003; Grimaldi-Bensouda
et al. 2016; Stasi et al. 1995). At the time of diagnosis, the patients who will experi-
ence spontaneous remission cannot be distinguished reliably from those who will
have chronic disease.
ITP is diagnosed clinically based on the presence of bleeding signs or symptoms
and isolated thrombocytopenia in the absence of other physical or laboratory abnor-
malities or other causes of thrombocytopenia (Kuhne et al. 2003). The bleeding
manifestations are variable but most often include mucocutaneous bleeding such as
bruising, petechiae, oral purpura, epistaxis, and menorrhagia (in females) (Kuhne
et al. 2011; Neunert et al. 2008). While the skin and mucous membrane findings can
be impressive, many children have surprisingly little bleeding despite profound
thrombocytopenia (Kuhne et al. 2003). While severe bleeding such as intracranial
hemorrhage is rare, it can occur unpredictably without evidence of trauma or other
C.M. Bennett, MD, MSc

Emory University School of Medicine, Atlanta, GA, USA
Aflac Cancer and Blood Disorder Center, Children’s Healthcare of Atlanta,
5461 Meridian Mark Road, Suite 400, Atlanta, GA 30342, USA

224 C.M. Bennett
inciting event and may cause serious morbidity and mortality (Kuhne et al. 2003;
Psaila et al. 2009).
The majority of children with ITP will have spontaneous disease resolution
within weeks to months of presentation (Imbach et al. 2006). About 20–30% of
children will have persistent thrombocytopenia beyond 6 months of diagnosis.
However, in contrast to adults with ITP, a significant proportion of children with
persistent ITP at 6 months will have disease resolution by 1 year (Imbach et al.
2006). The long-term remission rate is high, and in most children with chronic ITP,
the outcome is favorable (Rosthoj et al. 2012; Donato et al. 2009; Watts 2004;
Akbayram et al. 2011). Only about 5–10% of children who present with ITP will
have clinically significant chronic ITP that requires treatment (Bennett and Tarantino
2009; Aronis et al. 1994).
Since serious bleeding is unusual and spontaneous resolution common, children
with newly diagnosed ITP are often observed closely without treatment (Neunert
et al. 2011; Provan et al. 2010). Pharmacologic therapy can be reserved for patients
with significant or troublesome bleeding. Patients with chronic ITP who have mild
to moderate thrombocytopenia and minimal bleeding may be managed with close
observation alone or with standard first-line ITP therapies: corticosteroids, intrave-
nous immune globulin, or anti-D immune globulin (in patients who are Rh positive
and have intact spleens) (Neunert et al. 2011; Provan et al. 2010; Blanchette et al.
1994; Andrew et al. 1992; Papagianni et al. 2011; Blanchette et al. 1993; Imbach
et al. 1985). The small but significant group of children with severe thrombocytope-
nia and bleeding present a difficult therapeutic challenge. Second-line therapies
include splenectomy (Rijcken et al. 2014; Montalvo et al. 2014; Kuhne 2013),
immunosuppressive therapy, (rituximab, azathioprine, 6-mercaptopurine, mycophe-
nolate mofetil) (Rao et al. 2005; Provan et al. 2006; Saleh et al. 2000; Bennett et al.
2005; Sobota et al. 2009; Hilgartner et al. 1970), and thrombopoietin-receptor ago-
nists (eltrombopag, romiplostim) (Tarantino et al. 2016; Grainger et al. 2015). Only
eltrombopag and romiplostim are FDA approved for the treatment of chronic
ITP. While splenectomy is effective in children with ITP, it is often avoided in this
age group because of the lifelong risk of overwhelming bacterial sepsis (Ahmed
et al. 2016; Aronis et al. 2004; Kuhne et al. 2007). Immunosuppressive therapies
may be used as splenectomy sparing treatments but are not effective in many
patients. Thrombopoietin-receptor agonists may show higher efficacy, but long-
term treatment is usually necessary to maintain adequate platelet counts. There is no
standard of care for children with severe, symptomatic ITP, and treatment decisions
are usually based on patient preference, age, activity level, provider experience,
anecdotal evidence, expert opinion, and consensus guidelines.
While the long-term outcome for most children with ITP is favorable, during
periods of severe thrombocytopenia, there can be significant patient and family
anxiety about the ongoing risk of bleeding. Many children with chronic ITP have
activity restrictions which impact quality of life negatively. The ability to predict
disease course reliably would be helpful for families to manage anxiety and improve
quality of life. The identification of predictors of chronic disease in children with
ITP would be extremely beneficial to providers and families in making treatment
16 ITP in Childhood: Predictors of Disease Duration 225
decisions. Patients with a high likelihood of disease remission could be spared

unnecessary and potentially harmful treatment.
16.2 Challenges
The design of a prospective clinical trial with adequate power requires ample patient
numbers. Since chronic ITP is a rare disease, generating the numbers necessary for a
clinical trial at a single center is difficult. However, large multicenter studies are chal-
lenging and expensive to complete. Hence, the majority of studies investigating prog-
nostic factors in ITP are prospective observational studies or retrospective studies and
while these are valuable in forwarding our knowledge of the natural history of ITP,
they have limitations. A large proportion of patients in prospective long-term observa-
tional studies may be lost to follow-up, thereby limiting the analysis and subsequent
conclusions. In small single-center studies, the findings may not be generalizable to
all populations. In the many published ITP studies over the last decades, widely dis-
crepant criteria are used to evaluate patient characteristics, define chronic disease,
measure responses, and report outcomes. The resulting heterogeneity complicates the
interpretation of results and their application into clinical practice. The International
Working Group in ITP has recommended standardization of terminology for diagno-
sis and management of ITP with the goal of bringing harmonization to ITP research
(Rodeghiero et al. 2009). The new definitions for ITP diagnosis were based on the
high rate of spontaneous remission in children with ITP, even after 12 months. The
recommendations were to define three phases of ITP: newly diagnosed ITP (up to
3 months from diagnosis), persistent ITP (from 3–12 months), and chronic ITP (last-
ing more than 12 months). While this standardization may be helpful for future study
design and interpretation, it does not aid in comparing older studies of chronic ITP in
childhood to more recent ones. There is also heterogeneity in the parameters that are
studied. Predictors of interest in one study may not be included in the data collection
of another, so the results cannot be compared across studies. For example, in a recent
systemic review and meta-analysis of 54 articles published from 1975 to 2013, many
potentially interesting predictors such as antinuclear antibody titer were only reported
in a handful of studies and common baseline measurements such as mean presenting
platelet counts were only reported in 18 of 54 articles (Heitink-Polle et al. 2014).
Hopefully, with the development of new multicenter groups and the growth of estab-
lished registries, future studies will take a more harmonized approach. Despite these
challenges, the current body of evidence defining predictors of ITP outcome is valu-
able for disease management and patient quality of life.
16.3 Biomarker Predictors of Chronic ITP
The majority of recent studies that evaluate potential predictors of chronic disease
are retrospective or prospective observational studies. There is one recent ran-
domized controlled trial that investigated potential predictors of chronic disease at
226 C.M. Bennett
12 months after diagnosis. Table 16.1 summarizes the results of a selected list of

recent studies that have investigated predictors of chronic ITP.
In a multicenter, prospective study of children with newly diagnosed ITP con-
ducted by Nordic ITP study group, the following characteristics at diagnosis were
associated with chronic disease at 6 months post diagnosis: insidious onset of
symptoms (>2 weeks duration), older age (>10 years), no history of prior infec-
tion, absence of wet purpura, female gender, and platelet count >5 × 109/l (Zeller
et al. 2005). Insidious onset of disease was the strongest predictor of chronic
disease (Zeller et al. 2005). The Intercontinental Cooperative ITP Study Group
Registry I, a large multicenter, international, prospective observational study of
2540 children with newly diagnosed ITP, found that older age at diagnosis and
higher presenting platelet count increased the risk of chronic ITP at 6 months post
diagnosis (Kuhne et al. 2003). Infants under 1 year of age had the lowest likeli-
hood of chronicity (Kuhne et al. 2003). In the ICIS Registry II study, a prospective
observational study of 1239 children with newly diagnosed ITP, data were ana-
lyzed at 12 and 24 months to investigate factors that were associated with ITP
duration (Bennett et al. 2016). In a multivariate analysis of these data, younger
children, particularly children under 1 year of age were less likely to develop
chronic disease (Bennett et al. 2017). Gender, platelet count, and bleeding sever-
ity at diagnosis were not significant predictors of chronicity in the multivariate
analysis (Bennett et al. 2017).
Numerous retrospective studies have reported a significant associations
between chronic ITP (measured at 6 or 12 months) and various biomarkers, most
notably older age, insidious onset of symptoms, higher platelet count, negative
history of prior infection or immunization, the absence of mucosal bleeding, and
female gender, all measured at diagnosis (Donato et al. 2009; Akbayram et al.
2011; Chotsampancharoen et al. 2017; ElAlfy et al. 2010; Glanz et al. 2008). A
recent systematic review and meta-analysis reported that older age (>11 years),
higher platelet count (≥20 × 109/l), insidious onset of symptoms, no history of
prior infection or vaccination, and female gender were predictive of chronic ITP
(Heitink-Polle et al. 2014). Bleeding at diagnosis, a predictor of short ITP dura-
tion in some studies, was not significant in the systemic review (Heitink-Polle
et al. 2014).
Other laboratory findings have been studied less frequently but may be important
to include inclusion in future studies. Two studies have found significantly lower
absolute lymphocyte counts at diagnosis in patients developing chronic disease
(Deel et al. 2013; Ahmed et al. 2010). Antinuclear antibody testing has been studied
infrequently. In the meta-analysis and systemic review, there were three studies
which reported association between ANA positivity at diagnosis and chronic dis-
ease (Heitink-Polle et al. 2014). Other measures such as hemoglobin, platelet anti-
bodies, bone marrow results, and mean platelet volume did not consistently predict
chronic disease (Heitink-Polle et al. 2014).
Table 16.1 Selected list of studies evaluating predictors of chronic ITP in children
Patients
Duration Patients with Patients
of ITP enrolled outcome with cITP Features measured Significant
Year Type of study (months) n data n n (%) at diagnosis of ITP predictors of cITP Ref
2003 Prospective 6 2540 1742 651 (37.4) Age Older age (Kuhne et al. 2003)
Observational Gender Higher platelet
Multicenter Platelet count count (in children
Onset over 1 year of age
Prior infection only)
Bleeding Infants <1 year
have lowest risk of
cITP
2004 Prospective 6 506 423 106 (25.1) Age Insidious onset (Zeller et al. 2005)
Observational Gender Age ≥8 years
Multicenter Platelet count onset No prior infection
Prior infection Girls ages >7 years
Bleeding treatment with insidious
16 ITP in Childhood: Predictors of Disease Duration
Season onset of symptoms

have high risk of
cITP
2004 Prospective 6 63 60 16 (26.6) Age Higher platelet (Bruin et al. 2004)
Observational Gender count
Multicenter Platelet count (>10 × 109/l)
Prior infection No prior infection
Bleeding treatment FCGR2B 232I/T
Fcγ-receptor gene polymorphism
polymorphisms
(continued)
227
228
Patients
2006 Retrospective 6 79 72 19 (26.3) Age No prior infection (Roganovic and Letica-
Single center Gender insidious onset Crepulja 2006)
Platelet count
Onset of symptoms
bleeding
API
Season
2008 Retrospective 6 259 257 60 (23) Age Older age (Glanz et al. 2008)
Population-based Gender (>10 years)
Multicenter Platelet count Higher platelet
Prior infection count (>20 × 109/l)
Bleeding site No mucosal
Treatment bleeding
No prior infection
absence of
mucosal bleeding
Patients older than
10 years and with
platelets
>20 × 109/l had
highest risk of
chronic disease
C.M. Bennett
2009 Prospective 6 2605 1984 630 (31.8) Age Older age (Tamminga et al. 2009)
Matched pair Gender Higher platelet
Multicenter Platelet count (>20 × 109/l)
Prior infection No prior infection
Treatment Children treated
with IVIG were
less likely to have
chronic disease
2009 Retrospective 6 1683 1418 404 (28.5) Age Older age (Donato et al. 2009)
Multicenter Gender (>9 years)
Platelet count Higher platelet
Prior infection count
Bleeding severity (>10 × 109/l)
Type of purpura No prior infection
Treatment Infants have lowest
Seasonal incidence risk of chronic
disease
2010 Retrospective 6 409 344 120 (34.9) Age Older age (ElAlfy et al. 2010)
Multicenter Gender (>10 years)
Platelet count Higher platelet
Onset count (>20 × 109/l)
Prior infection Insidious onset
Bleeding severity No mucosal
Bleeding site bleeding
Treatment No prior infection
(continued)
229
230
Patients
2010 Retrospective 6 625 475 270 (56.8) Age Older age (Bansal et al. 2010)
Multicenter Gender (≥8 years)
Platelet count Male gender
Bleeding site
Bleeding severity
2011 Retrospective 6 260 260 69 Age Older age (Akbayram et al. 2011)
Single center Gender (>10 years)
Platelet count No prior infection
Onset Higher platelet
Prior infection count
Treatment
Season
Bleeding
2013 Retrospective 3 472 312 Not Age Older age (Revel-Vilk et al. 2013)
6 reported Gender (≥10 years)
12 Platelet count Insidious onset
Onset
Prior infection
Bleeding
Treatment
C.M. Bennett
2014 Systemic review 6 N/A N/A N/A Age Older age (Heitink-Polle et al. 2014)
& 12 Gender (≥11 years)
Meta-analysis Platelet count Female gender
Prior infection Higher platelet
Onset count
Bleeding No prior infection
Treatment Insidious onset
Hemoglobin ANA positivity
White blood cells Treatment with
Antinuclear corticosteroids and
antibody (ANA) IVIG
Platelet antibody Treatment with
Bone marrow IVIG alone was
parameters protective
Genetic factors
2016 Prospective 12 1239 705a 286a Age Older age (Bennett et al. 2016)
Observational 24 383b 152b Gender (≥10 years)
Platelet counts Less bleeding

Bleeding severity (significant at
Treatment 12 months only)
Combination
therapy with IVIG
and corticosteroids
protective
(continued)
231
Patients
232

2016 Randomized 12 100 patients 10.4 Age Age (Heitink-Polle et al. 2016)
controlled trial observation only Gender (>10 years)
(2009–2015) 100 patients 10.2 Platelet count Insidious onset
IVIG Onset No mucosal
Prior infection bleeding
Bleeding Lower leukocyte
Leukocyte count count
Lymphocyte count Lower lymphocyte
Treatment response count
Absence of
complete response
to IVIG at 1 week
was associated
with cITP
cITP not lower in
IVIG arm
2017 Retrospective 12 417 405 113 Age Older age (Chotsampancharoen et al.
Single center Gender (>5 years) 2017)
Platelet count Insidious onset
Treatment Platelet count at
Onset of symptoms 4 weeks post
Bleeding diagnosis of at
Prior infection least ≥100 × 109/l
was associated ITP
resolution by
12 months
cITP chronic ITP
a
C.M. Bennett
705 patients had outcome data at 12 months; 286 of these had cITP
b
383 patients had outcome data at 24 months; 152 of these had cITP
16.4 Treatment-Related Factors
Management of children with newly diagnosed ITP consists of close observation ver-
sus treatment with IVIG or corticosteroids. Two recent guidelines support the use of
careful observation over treatment in asymptomatic children with newly diagnosed
ITP (Neunert et al. 2011; Provan et al. 2010). However, several observational and
retrospective studies suggested a lower risk of chronic ITP in children treated with
IVIG. In a matched pair analysis of the ICIS I Registry data, children initially treated
with IVIG were more likely to have ITP resolution than children who received no
therapy (Tamminga et al. 2009). In the meta-analysis, 14 studies assessed the associa-
tion between initial treatment with IVIG and chronic disease (Heitink-Polle et al.
2014). This analysis showed significantly less chronic ITP in patients treated with
IVIG at diagnosis (Heitink-Polle et al. 2014). In the ICIS Registry II study, treatment
with IVIG at diagnosis was not protective against the development of chronic ITP, but
interestingly, combination therapy with IVIG and corticosteroids was associated with
a lower risk of chronic disease (Bennett et al. 2017). In contrast, the meta-analysis
reported a higher risk of chronic ITP in patients who received a combination of IVIG
and methylprednisolone at diagnosis (Heitink-Polle et al. 2014).
The initial findings of a phase 3, multicenter, randomized, controlled clinical trial
evaluating the efficacy of IVIG treatment versus close observation in children with
newly diagnosed ITP were presented at the annual meeting of the American Society
of Hematology in 2016 and appear to have settled this issue. In this study, the rate
of chronic ITP did not differ significantly between the IVIG treated group and the
observation only group (Heitink-Polle et al. 2016). However, in patients random-
ized to receive IVIG, recovery rates were higher in the first 3 months after diagno-
sis, and bleeding was less common than in the observation group (Heitink-Polle
et al. 2016). The study also investigated biomarker predictors of chronic disease and
found that the following features at diagnosis were associated with a higher risk of
developing chronic disease: older age, longer duration of symptoms, no mucosal
bleeding, lower leukocyte count, and lower lymphocyte count (Heitink-Polle et al.
2016). This study supports prior retrospective studies showing the significance of
lymphocyte and leukocyte counts in the development of chronic ITP (see above). In
the IVIG group, the absence of complete response at 1 week was associated with
development of chronic ITP (Heitink-Polle et al. 2016).
16.5 Genetic Factors
With the development of modern genetic methods, novel approaches are being used
to identify markers of disease outcome and potential therapeutic targets. Several
studies have utilized genetic approaches to investigate factors that might be involved
in the pathogenesis of ITP and might predict outcome or treatment response.
In a gene expression profile of whole blood samples of patients with newly diag-
nosed and chronic ITP, children with chronic disease showed significant upregula-
tion of interferon-regulated genes (Sood et al. 2008) and overexpression of VNN1, a
gene involved in the oxidative stress pathway (Zhang et al. 2011).
234 C.M. Bennett
Functional variants of inflammatory cytokine genes and low-affinity Fcγ recep-

tor (FcγR) genes have been implicated in autoimmune disease. In a pilot study of
37 children with chronic ITP and 218 controls, genotyping was performed on
common variants in the regulatory regions of cytokines (TNF, LTA, IL1RN, IL1A,
IL4, IL6, IL10) and structural variants of the low-affinity FcγRs (FCGR2A,
FCGR3A, FCGR3B) (Foster et al. 2001). Two combinations of genotypes, TNF
and FCGR3A, were significantly associated with chronic ITP (Foster et al. 2001).
In a prospective study of newly diagnosed ITP patients, the FCGR2B-232I/T
polymorphism was overrepresented in patients who developed chronic disease
(Bruin et al. 2004). In the systemic review and meta-analysis of predictors of
chronic ITP, 18 articles reported allele frequencies of 29 different genes, but only
two allele frequencies showed significantly higher risks for chronic ITP: TGF-B1
cod 25 allele A and IL-4 intron 3 allele RP1 (Heitink-Polle et al. 2014). In recent
study using whole genome sequencing of genes associated with cellular immunity
in patients with chronic ITP, two genes IFNA17 (interferon alpha 17) and IFNLR1
(interferon-lambda receptor 1) which are involved in T-cell function showed an
increased frequency in children with chronic ITP compared to controls (Despotovic
et al. 2015).
In a 2010 review of genetic studies in pediatric ITP, Bergmann et al. assessed the
feasibility, advantages and disadvantages of potential genetic approaches in study-
ing pediatric ITP including linkage studies, genome-wide association studies, com-
parative genomics, candidate gene approaches, and pharmacogenetics studies and
reviewed the current literature of genetic factors associated with pediatric ITP
(Bergmann et al. 2010). This report may be referenced for a more in-depth review
of genetic studies in childhood ITP that is beyond the scope of this chapter.
16.6 Discussion
ITP in children is typically a self-limited disorder that resolves within several weeks
to months after the onset of symptoms. While the risk of clinically significant bleed-
ing is low and the outcome is favorable in the majority of patients, children with
severe thrombocytopenia require close observation and activity restrictions, which
often contribute to patient and parental anxiety and negatively impact quality of life.
Chronic ITP in childhood has a benign course in the majority of cases and may
resolve with or without therapy even several years from onset (Aronis et al. 1994).
A small but significant group of patients will have severe, chronic ITP that requires
ongoing treatment to prevent or treat bleeding. At diagnosis, it is not possible to
distinguish patients who will have a short ITP duration from those who will have
chronic disease. Identifying reliable prognostic factors at diagnosis that predict ITP
outcome would be invaluable for clinicians and families to guide treatment deci-
sions and ultimately improve quality of life.
Despite the limitations of current research, there is a considerable body of
evidence to support these predictors of chronic ITP in children which are present
at initial diagnosis: older age, higher platelet count, longer duration of
symptoms, female gender, absence of a preceding infection, lower leukocyte

count, and lower lymphocyte count. However, in a randomized clinical trial mea-
suring the effect of IVIG treatment on the development of chronic disease, female
gender, higher platelet count, and the absence of a prior infection or immuniza-
tion were not significant predictive factors. Therefore, based on this new evi-
dence, these biomarkers may not be strongly predictive of chronic disease in
childhood ITP after all.
Several prior studies demonstrated a potentially protective effect of IVIG treat-
ment at diagnosis against the development of chronic ITP (Blanchette et al. 1993;
Tamminga et al. 2009). These findings were interesting and had potential clinical
significance in the treatment of newly diagnosed children. The recent randomized,
controlled clinical trial showed that the rate of chronic ITP was no different in the
IVIG group compared to observation alone. However, this study did show that in the
IVIG group, recovery rates were higher during the first 3 months, and bleeding was
less frequent than in the observation group.
The prospective observational ICIS II registry study indicated that combination
therapy with both IVIG and corticosteroids led to a lower risk of chronic ITP, but the
large number of patients lost to follow-up was problematic in this study and may
have impacted the analysis. By contrast, in a meta-analysis and systematic review, a
significantly higher risk for chronic ITP was found in patients treated with IVIG and
corticosteroids in combination, but only a univariate analysis could be performed,
so confounding and bias may have influenced the results.
Based on the current somewhat contradictory evidence, it would be inappropriate
to recommend up front pharmacologic therapy to young children with newly diag-
nosed ITP solely to prevent the development of chronic disease, given that most
children have spontaneous disease resolution and no clinically significant bleeding.
However, treatment with IVIG alone or in combination with corticosteroids might
be beneficial to selected patients with clinically significant bleeding.
Various candidate genes and genetic pathways have been implicated in the devel-
opment of autoimmune disease, including ITP, but to date, there is no convincing
reproducible evidence for any genetic markers of ITP outcome. Large multicenter
studies are necessary to discover genes or genetic pathways involved in the develop-
ment of chronic ITP.
Conclusions
While great progress has been made in discovering risk factors which determine
ITP outcome, important questions and challenges remain. The current predictors
present at ITP diagnosis, specifically younger age, shorter duration of symptoms,
higher leukocyte count, and higher lymphocyte count, can be applied to newly
diagnosed patients to help determine the likelihood of short disease duration, and
these patients can be managed with close observation and treated only if bleed-
ing. Unfortunately, the current predictors are not adequate in identifying those
patients who will ultimately develop clinically significant chronic disease. Future
studies utilizing large ITP registries and genetic approaches are needed to dis-
cover novel biomarkers that reliably predict ITP course.
236 C.M. Bennett
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Immune Functions of Platelets
17
Rick Kapur and John W. Semple
17.1 General Introduction
Platelets are anucleate and small cellular fragments (~2–4 μM in diameter) derived
from bone marrow-resident megakaryocytes (~50–100 μM in diameter) and are
known to be indispensable for the regulation of hemostasis (Semple et al. 2011).
Platelet generation is a complex and highly regulated process (Machlus and
Italiano 2013), and recently it has been suggested that also the lung may be an
important site for the production of platelets (Lefrancais et al. 2017). This may
perhaps reflect the diverse nature of platelets, and indeed a recent body of work
has revealed that platelets are involved in several other functional processes
beyond hemostasis, in both health and disease (Kapur et al. 2015b; Semple et al.
2011; Youssefian et al. 2002; McMorran et al. 2009; Wong et al. 2013; Clark et al.
2007; Sreeramkumar et al. 2014; Boilard et al. 2010; Kapur and Semple 2016a,
b). These non-hemostatic functions will be discussed in this chapter generally
outlined as “Platelets and Pathogens,” reflecting the ability of platelets to battle
R. Kapur
Division of Hematology and Transfusion Medicine, Lund University,
BMC C14, Klinikgatan 26, Lund 221 84, Sweden
J.W. Semple (*)
Division of Hematology and Transfusion Medicine, Lund University,
BMC C14, Klinikgatan 26, Lund 221 84, Sweden
Keenan Research Centre for Biomedical Science, St. Michael’s Hospital,
Toronto, ON, Canada
The Toronto Platelet Immunobiology Group, St. Michael’s Hospital, Toronto, ON, Canada
Canadian Blood Services, Toronto, ON, Canada
Departments of Pharmacology, Medicine, and Laboratory Medicine and Pathobiology,

242 R. Kapur and J.W. Semple
invading pathogens, and “Platelet-Target Cell Communication,” illustrating how

platelets use sophisticated channels in order to communicate with a variety of
effector cells.
17.2 Platelets and Pathogens
17.2.1 Pathogen Recognition
Platelets initiate their antimicrobial host defense through sensing the threat of
invading pathogens or damage during inflammation, via their immune receptors
called pattern recognition receptors. These include immunoglobulin or comple-
ment receptors and Toll-like receptors (TLRs) (Semple et al. 2011). These recep-
tors directly bind invading pathogens and microbes, including their derived
materials. Pathogens first encounter TLRs on professional phagocytes, such as
neutrophils, dendritic cells (DCs), or macrophages (Semple et al. 2011; Janeway
1992; Janeway and Medzhitov 2002). The expression of TLRs 1–9 has been
described on human as well as on murine platelets, demonstrating a functional
role for some of these TLRs (Semple et al. 2011). For instance, TLR4 was shown
to mediate lipopolysaccharide (LPS, a gram-negative endotoxin)-induced throm-
bocytopenia and TNF-α production in vivo (Andonegui et al. 2005; Cognasse
et al. 2005; Aslam et al. 2006; Semple et al. 2007; Patrignani et al. 2006; Stahl
et al. 2006; Zhang et al. 2009a). Also, TLR4-knockout mice displayed decreased
circulating platelet counts and reticulated platelets, suggesting TLR4 to be of
importance in platelet production (Andonegui et al. 2005; Jayachandran et al.
2007). Triggering of TLR2 on human platelets by Pam3CSK4, a synthetic ligand
mimicking bacterial lipopeptide, enabled a thromboinflammatory response via
activation of phosphoinositide 3-kinase (Blair et al. 2009). Platelet TLR2 and
TLR4 were also involved in a setting of periodontitis, which is associated with
an increased risk for cardiovascular diseases (Assinger et al. 2012). The peri-
odontopathogens (A. actinomycetemcomitans and P. gingivalis) upregulated the
expression of CD40L, known to mediate thrombotic and inflammatory processes,
on human platelets via TLR2 and TLR4 (Assinger et al. 2012). Additionally,
platelet TLR3 was shown to respond to poly I:C, indicating an effect on innate
immune responses when detecting viral dsRNA (Anabel et al. 2014). Platelet
TLR7, on the other hand, mediated host survival and platelet counts during infec-
tion with encephalomyocarditis virus (EMCV) in mice, independently of throm-
bosis (Koupenova et al. 2014). In contrast to the sensing of external danger
signals, platelet TLR9 appears to be more important as a sensor for internal sig-
nals. It was shown that platelet TLR9 was functional in a setting of oxidative
stress, through stimulation of platelet activation, granule secretion, aggregation
in vitro, and thrombosis in vivo (Panigrahi et al. 2013). Notably, thrombocytope-
nia was shown to impair the host defense during pulmonary infection with the
gram-positive bacteria S. pneumoniae in mice (van den Boogaard et al. 2015);
17 Immune Functions of Platelets 243
however, platelet activation by S. pneumoniae was subsequently shown to occur

independently of TLR signaling (de Stoppelaar et al. 2016), indicating a different
recognition mechanism of S. pneumoniae by platelets. Taken together, besides
their hemostatic functions, platelets play a role as pathogen sensors in the blood
circulation.
17.2.2 Pathogen Retainment
Platelets can harbor pathogens on their plasma membrane as well as internally

(Youssefian et al. 2002; Flaujac et al. 2010), such as viruses (Assinger 2014; Flaujac
et al. 2010), bacteria (Yeaman 2010a, b; Kerrigan and Cox 2010), and parasites
(McMorran et al. 2009). Platelets were also involved in acute and chronic hepatic
disease due to hepatitis B virus, via upregulating virus-specific CD8+ T cells and
nonspecific inflammatory cells into the liver (Aiolfi and Sitia 2015). Interestingly,
activated platelets were shown to surround or encapsulate the bacterium
Staphylococcus aureus, forcing the pathogens into clusters resulting in restricted
bacterial growth (Kraemer et al. 2011). This mechanism was dependent upon secre-
tion of the antimicrobial peptide β-defensin and signaling through neutrophil extra-
cellular trap (NET) formation (Kraemer et al. 2011). NETs have now been suggested
to be involved in both infectious and noninfectious pathologies including thrombo-
sis and coagulopathy, tumor growth, cardiac fibrosis, transfusion-related acute lung
injury, sickle cell disease, storage of red blood cells, and diabetes (Fuchs et al. 2010;
Thomas et al. 2012; Demers et al. 2012; Fuchs et al. 2013; Chen et al. 2014;
Caudrillier et al. 2012; Wong et al. 2015; Martinod et al. 2016; Demers et al. 2016;
Martinod et al. 2017; Jorch and Kubes 2017). Bacteria (methicillin-resistant
S. aureus and Bacillus cereus) were also trapped on hepatic Kupffer cells, via engag-
ing platelet adhesion receptor GP1b (Wong et al. 2013). In that study, infected
GP1bα-deficient mice suffered more endothelial cell and Kupffer cell damage,
resulting in more vascular leakage and rapid mortality (Wong et al. 2013). Activation
of platelets during sepsis can contribute to disseminated intravascular coagulation,
resulting in blood vessel occlusion, increased ischemia, and multiple organ failure,
and it can also contribute to stimulation of pro- and anti-inflammatory cytokine
production (de Stoppelaar et al., de Stoppelaar et al. 2014). This platelet activation
is evident from increased surface P-selectin expression (Gawaz et al. 1997;
Russwurm et al. 2002) and increased levels of triggering receptor expressed on
myeloid cell-like transcript-1 (Washington et al. 2009) or PF-4 in mice (de
Stoppelaar et al. 2013). Sepsis patients with platelet counts <50 × 109/L, however,
displayed a dysregulated host response which included increased endothelial cell
activation without differences in coagulation activation (Claushuis et al. 2016). In
addition, during sepsis neutrophils were also shown to be activated by platelet
TLR4, enabling the release of NETs, which trapped bacteria in blood vessels of the
liver and lungs (Clark et al. 2007). It was suggested that platelets can act as circula-
tory sentinel cells, sensing infectious agents and presenting them to neutrophils
and/or the reticuloendothelial system (Aslam et al. 2006; Semple et al. 2007;
Patrignani et al. 2006; Stahl et al. 2006; Zhang et al. 2009a). On one hand platelet-
dependent NET formation may have its beneficial effects as bacteria are entangled
in NETs; on the other hand, it may also be detrimental to the host. When neutrophils
are activated by LPS and come into contact with the endothelium, there is little
injury; however, if bound neutrophils encounter platelets with bound LPS, neutro-
phils are activated resulting in NET formation accompanied by release of reactive
oxygen species, which damages the underlying endothelium (Clark et al. 2007).
Furthermore, it has been reported that neutrophils are able to scan platelets for acti-
vation in the bloodstream via P-selectin ligand signaling, enhancing inflammation
(Sreeramkumar et al. 2014). Platelet P-selectin, soluble or cellular, was also
described to stimulate NET formation in mice through binding to neutrophil
P-selectin glycoprotein ligand-1 (PSGL-1) (Etulain et al. 2015). Also in a setting of
diabetes, in which neutrophils are more susceptible to NET formation, NETs were
found to impair wound healing. It was therefore suggested that cleaving NETs or
inhibiting their formation may be an effective approach to improve wound healing
and reduce inflammation in diabetes (Wong et al. 2015).
17.2.3 Pathogen Elimination
Platelets have not only been implicated in detecting and retaining bacteria; they
also appear to be involved in the clearance of bacterial infections. For instance,
in infective endocarditis, thrombin-stimulated platelets were shown to facilitate
the clearance of streptococci (Dankert et al. 1995). Additionally, in murine
P. gingivalis infection, platelet TLR2 was implicated in the generation of plate-
let-neutrophil aggregates (Blair et al. 2009), and later it was demonstrated that
phagocytosis of periodontopathogens by neutrophils occurred via platelets,
TLR2, and plasma factors (Assinger et al. 2011). Also, platelets could redirect
the course of the bacteria Listeria monocytogenes from less immunogenic phago-
cytes toward the more immunologically active CD8α+ dendritic cells in the
spleen, using a mechanism dependent upon GP1b and complement component
C3 (Verschoor et al. 2011). Furthermore, McMorran and colleagues elegantly
demonstrated that activated platelets can kill the malarial parasite Plasmodium
inside the red blood cell (McMorran et al. 2009). In a follow-up study, they elu-
cidated that this platelet-mediated killing of Plasmodium was dependent upon
platelet factor 4 (PF4 or CXCL4) and the erythrocyte Duffy-antigen receptor
(Fy) (McMorran et al. 2012). This implies that Duffy-negative individuals, thus
lacking Fy, would be incapable of eliminating this intraerythrocytic malarial
parasite through activated platelets. In apparent contrast, however, a recent study
reported that there is no evidence to support Plasmodium killing by platelets and
that platelets do not contribute to a protective immune response which clears the
Plasmodium infection, but that they activate a pathogenic response to Plasmodium
using platelet CD40 (Gramaglia et al. 2017).
17.2.4 Pathogen Evasion
As a countermeasure, viruses and bacteria are able to evade these immune

responses elicited by platelets. This can be supported by the fact that acute viral
or bacterial infections often result in low platelet counts or thrombocytopenia.
This has frequently been observed in the autoimmune bleeding disorder immune
thrombocytopenia (ITP) (Cines et al. 2014). The pathogenesis of infection-
induced platelet destruction is incompletely understood, but several mechanisms
have been suggested. These include molecular mimicry between platelet anti-
gens and viral/bacterial antigens, which triggers cross-reactive autoantibody pro-
duction (Zhang et al. 2009b; Wright et al. 1996; Takahashi et al. 2004; Li et al.
2005; Chia et al. 1998). In addition, ITP patients infected with the gram-negative
bacteria Helicobacter pylori displayed an elevation of their platelet counts fol-
lowing Helicobacter pylori-eradication therapy (Asahi et al. 2008). Similarly,
the gram-negative bacterial endotoxin LPS also enhanced antiplatelet antibody-
mediated phagocytosis of platelets in vitro (Semple et al. 2007), as well as an
increased platelet clearance in vivo upon coinjection of antiplatelet antibodies
and LPS in mice (Tremblay et al. 2007). Furthermore, C-reactive protein (CRP),
an acute phase protein which rapidly increases during acute bacterial and viral
infections and therefore is clinically used as a biomarker for acute infections and
inflammation, was found to strongly enhance antibody-mediated platelet destruc-
tion both in vitro and in vivo in mice (Kapur et al. 2015a). CRP was also found
to be elevated in pediatric ITP, and treatment with IVIg was correlated with
increased platelet counts, decreased levels of CRP, and reduced clinical bleeding
severity (Kapur et al. 2015a). Interestingly, an increased CRP value at diagnosis
appeared to be predictive for slower platelet count recovery after 3 months
(Kapur et al. 2015a). This finding was validated in a cohort of newly diagnosed
adult ITP patients, in which increased CRP levels at diagnosis were shown to
negatively predict recovery of platelet counts after steroid treatment (Rama
Kishore et al. 2017).
17.3 Platelet-Target Cell Communication
17.3.1 Release of Mediators
Platelets can also elicit immune response through release of several mediators
including platelet CD40L, which is released upon platelet activation giving rise
to soluble CD40L (sCD40L) in circulation (Henn et al. 2001). Platelet-derived
CD40L can also enhance CD8+ T-cell responses upon infection with Listeria
monocytogenes (Elzey et al. 2008; Iannacone et al. 2005) and bind to dendritic
cells (DCs) impairing differentiation, suppressing proinflammatory DC cyto-
kines (IL-12p70 and TNF), and increasing the production of the anti-inflamma-
tory cytokine IL-10 by DCs (Kissel et al. 2006). Furthermore, activated platelets
were described to enhance lymphocyte adhesion to endothelial cells (Diacovo

et al. 1998) and facilitate lymphocyte homing in high endothelial venules
(Diacovo et al. 1996). Additionally, platelets are able to stimulate B-cell differ-
entiation and Ab class switching via their CD40L (von Hundelshausen and
Weber 2007; Elzey et al. 2003). Several other signaling pathways have been
linked to platelet activation via the CD40L-CD40 axis, including NF-κB
(Hachem et al. 2012; Malaver et al. 2009; Spinelli et al. 2010; Gambaryan et al.
2010; Karim et al. 2013; Liu et al. 2002), illustrating that platelets are well
equipped for modulating adaptive immune responses via their CD40L and/or
their secreted sCD40L.
Platelets can secrete a multitude of different cytokines and chemokines, most
of them located within different granules, which may differently impact hemo-
stasis and wound repair on one hand (Mazzucco et al. 2010), but they may also
affect proinflammatory or anti-inflammatory immune reactions on the other hand
(Assoian et al. 1983). TGF-β levels, for instance, seem regulated by platelets in
an autoimmune setting of ITP, as low levels of TGF-β were observed during
active ITP while those levels normalized again upon successful treatment of ITP
which increased the platelet counts and normalized the T-regulatory cell numbers
(Andersson et al. 2000, 2002). The platelet α granules contain a large variety of
soluble immune factors, like chemokines, which include PF (CXCL4), RANTES
(CCL5), β-thromboglobulin (β-TG, an isoform of CXCL7), and MIP-1α (CCL3)
(Blair and Flaumenhaft 2009). These chemokines are released upon platelet acti-
vation and elicit diverse immunomodulatory responses. PF-4, for example, ren-
ders monocytes resistant to apoptosis and enhances their differentiation into
macrophages (Gleissner 2012). Besides that, PF-4 can stimulate neutrophil adhe-
sion to unstimulated endothelial cells and release content from platelet granules
(Petersen et al. 1999). In contrast, platelet-derived β-TGs, which are proteolytic
products of inactive precursors, can either stimulate or inhibit neutrophil activity
(Brandt et al. 2000). On the other hand, PF4 can negatively regulate Th17 dif-
ferentiation, thereby limiting murine cardiac allograft rejection (Shi et al. 2014a).
Also, platelet-derived MIP-1α can enhance histamine release from basophils
(Alam et al. 1992) and is chemotactic for T cells (Schall et al. 1993). In addition,
platelet can also release IL-33 which was found to induce eosinophilic airway
inflammation (Takeda et al. 2016).
17.3.2 Microparticle Shedding
Platelet microparticles (also referred to as microvesicles) are small extracellular

vesicles produced by budding of the cytoplasmic membrane. Originally, they
were described as “dust” released from activated platelets which supported
thrombin generation, even without the presence of intact platelets (Wolf 1967).
Generally, the size of microparticles ranges from ~100 to 1000 nm in diameter,
although the majority are ~200 nm. Microparticles are distinct from exosomes,
which are ~50–100 nm in diameter and originating via exocytosis from multive-
sicular bodies (Buzas et al. 2014). Platelets are potent producers of microparti-
cles, as compared to other cell types, as was demonstrated by the high abundance
of platelet microparticles in circulation using cryotransmission electron micros-
copy and gold nanospheres conjugated to antibodies against the platelet CD41
(Arraud et al. 2014). Microparticle formation is associated with increased levels
of intracellular calcium, cytoskeletal rearrangement, and membrane phosphati-
dylserine (PS) exposure (Morel et al. 2011), which supports coagulation
considering its anionic properties, but platelet microparticles express modest
levels of tissue factor (TF) and seem to be less procoagulant than their mono-
cyte-derived counterparts, which express PS as well as TF (Owens and Mackman
2011). The proteasome also appears to be important for microparticle forma-
tion, as the proteasome inhibitor bortezomib was found to reduce platelet mic-
roparticle release following stimulation with thrombin, LPS, or ADP (Gupta
et al. 2014). Examination of platelet activation under physiological flow condi-
tions revealed elongated membrane tendrils (up to 250 μM) emerging from
platelets, so-called flow-induced protrusions (FLIPRs) (Tersteeg et al. 2014).
FLIPRs also expose PS, recruit monocytes and neutrophils, and appear to shed
off PS+ microparticles (Tersteeg et al. 2014). In contrast, however, PS- mic-
roparticles have also been described in body fluids (Arraud et al. 2014; Connor
et al. 2010; Perez-Pujol et al. 2007; Cloutier et al. 2013), demonstrating the
complexity of platelet microparticle production. Platelet microparticles have
been described in various inflammatory conditions, in which platelets become
activated (Nurden 2011; Reid and Webster 2012), and clinically they may be
associated with disease progression. For example, in blood and synovial fluid of
patients suffering from rheumatoid arthritis (RA), platelet microparticles were
found to be elevated (Boilard et al. 2010; Gyorgy et al. 2012; Rousseau et al.
2015; Gitz et al. 2014; Boilard et al. 2012). Moreover, in vivo depletion of plate-
lets in a murine RA model was shown to attenuate the inflammation (Boilard
et al. 2010; Mott and Lazarus 2013). As microparticles are observed in sterile,
as well as under inflammatory, conditions, it remains unclear how exactly plate-
let microparticle formation is regulated. Multiple signaling pathways may be
leading up to platelet activation and triggering the production of platelet mic-
roparticles, such as via apoptosis, high shear forces, or platelet surface recep-
tors. The disease setting, at least partly, influences how microparticles are
shedded, as in RA the collagen receptor glycoprotein VI (GPVI) becomes acti-
vated, while in sepsis microparticles are produced via TLR4 signaling via LPS
(Boilard et al. 2010; Brown and McIntyre 2011). Both these signals, however,
are accompanied by increased IL-1 levels, indicating a common proinflamma-
tory source. Additionally, microparticles can be formed by signaling through
immune complexes, made up of bacterial components and well-conserved epit-
opes expressed by influenza viruses, engaging the platelet FcγRIIA (Boilard
et al. 2014; Sun et al. 2013).
From a functional perspective, platelet microparticles can facilitate communica-

tion of platelets with other cells and thereby regulate immune functions, as platelet
microparticles can contain a heterogeneous cargo consisting of various cytokines
and chemokines (e.g., IL-1, RANTES), potent lipid mediators (e.g., thromboxane
A2), enzymes (e.g., inducible NO synthase), surface receptors (e.g., CD40L), auto-
antigens (e.g., citrullinated fibrinogen), nucleic acids (e.g., microRNA), transcrip-
tion factors (e.g., PPARγ, RuvB-like2, STAT3, STAT5a), or respiratory competent
mitochondria (Laffont et al. 2013; Cloutier et al. 2013; Nurden 2011; Reid and
Webster 2012; Boudreau et al. 2014; Ray et al. 2008; Garcia et al. 2005). As the
microparticles can express PS and surface receptors, they interact with other cells
via integrin and through the PS-binding proteins lactadherin (Dasgupta et al. 2009)
and developmental endothelial locus-1 (Del-1) (Dasgupta et al. 2012). These pro-
teins appear to be involved in the clearance of microparticles and the interaction
with other cells, as Del-1 −/− and lactadherin −/− mice appear to have a high
degree of microparticles in their plasma (Dasgupta et al. 2009, 2012). Transcription
factors transported within platelet microparticles can enable transcellular effects,
such as PPARγ, which was packed inside platelet microparticles and transferred to
monocytes where it conveyed transcellular effects (Ray et al. 2008). Platelet-derived
microparticles were also shown to inhibit IL-17 and IFN-γ production by
T-regulatory cells (Tregs) and stimulate Treg stability during inflammation, in a
P-selectin-dependent manner (Dinkla et al. 2016). Currently, however, more
research is required to establish the nature of the specific signals which lead to the
internalization of microparticles by the recipient target cells. Microparticles appear
to be important biomarkers in inflammatory disorders, but further delineation of
their function, mechanisms of generation, and routing are warranted, in order to
better understand their role in health and disease (Melki et al. 2017).
17.3.3 RNA Transfer
Platelets are known to express and secrete many different molecules during
platelet activation, and they do so via distinct mechanisms (Blair and Flaumenhaft
2009; Italiano et al. 2008; Sehgal and Storrie 2007; White and Rompietti 2007).
Despite being anucleate, platelets have been shown to express significant amounts
of RNA, including mRNAs (e.g., (pre)mature RNA), structural and catalytic
RNAs (e.g., ribosomal and tRNA), regulatory RNAs (e.g., microRNA), and non-
coding RNA (e.g., antisense RNA) (Rowley et al. 2011, 2012; Lood et al. 2010;
Healy et al. 2006; Goodall et al. 2010; Simon et al. 2014; Edelstein et al. 2013;
Ple et al. 2012; McManus et al. 2013; Freedman et al. 2010; Raghavachari et al.
2007; Risitano et al. 2012; Clancy and Freedman 2014; Laffont et al. 2013;
Gidlof et al. 2013; Landry et al. 2009; Rondina and Weyrich 2015). Moreover,
platelets also possess the molecular machinery which allows them to translate
mRNA into proteins and to transfer RNA to recipient cells, such as platelet
microRNA-223 which is transferred to human umbilical vein endothelial cells

(Risitano et al. 2012; Clancy and Freedman 2014; Laffont et al. 2013; Gidlof
et al. 2013; Rondina and Weyrich 2015). Intercellular transfer of platelet RNA to
target cells can occur via platelet microparticles, as described earlier. However,
the content of platelet RNA transcript does not fully match to the platelet pro-
teome content (Burkhart et al. 2012). The role of platelet mRNA and its impact
on platelet function in both health and disease is currently actively being investi-
gated (Schubert et al. 2014).
17.3.4 Platelet MHC Class I Signaling
Platelets harbor two different types of MHC class I molecules: platelet plasma
membrane bound and intracellularly (Shulman et al. 1962). The MHC class I
molecules on the platelet plasma membrane are mainly adsorbed from plasma.
The platelet-membrane MHC class I molecules appear to be somewhat instable,
as can passively dissociate from the platelet during storage or can be eluted
from the membrane upon chloroquine diphosphate or acid treatment, without
affecting the platelet-membrane integrity (Blumberg et al. 1984; Kao et al.
1986; Kao 1987, 1988; Neumuller et al. 1993; Ghio et al. 1999; Gouttefangeas
et al. 2000). Interestingly, denatured MHC class I can elicit faulty interactions
with CD8+ T cells, energizing CTLs, following transfusions. For instance, allo-
geneic platelet MHC class I molecules are incapable of stimulating CTL-
mediated cytotoxicity on their own (Gouttefangeas et al. 2000) but can mediate
an immunosuppressive-like reaction to transfused blood cells. CBA mice trans-
fused with allogeneic BALB/c platelets accepted donor-specific skin grafts, in
contrast to non-transfused recipients (Aslam et al. 2008). This implies that allo-
geneic platelets may inhibit T-cell-mediated cytotoxicity reactions, like skin
graft rejections. Intracellular platelet MHC class I, on the other hand, is associ-
ated with α granules and generally consists of intact integral membrane proteins
associated with β2-microglobulin (Zufferey et al. 2014). It was also demon-
strated that platelets contain the entire proteasome system, including TAP mol-
ecules, but the endoplasmic reticulum is absent. Interestingly, the proteasome
function was found to be critical for thrombopoiesis (Shi et al. 2014b). In syn-
geneic settings, platelet activation can lead to expression of nascent MHC class
I molecules, which are capable of presenting antigens to CD8+ T cells. Activated
platelets were shown to present malarial peptides to malaria-specific T cells,
resulting in enhanced immunity against the parasite (Chapman et al. 2012).
Therefore, the type of platelet MHC class I, bound to the platelet plasma mem-
brane or intracellularly, will determine the response toward T cells (suppression
or activation).
A brief summary of the immune-sensing functions of platelets is depicted in
Fig. 17.1.
Target Cell Communication

PLATELETS • Release of CD40L upon platelet
activation:
Immune
Sensing - T cell triggering upon infection
with L. monocytogenes
Functions
- DC binding and impairment
DC-differentiation: shifting
cytokine secretion from pro- to
anti-inflammation
Pathogen Targeting - Stimulation of B cell differentiation

and antibody class switching
• Pathogen recognition
Via surface immune receptors • Secretion of cytokines and chemo-
• Pathogen capture kines:
On plasma membrane or Pro/anti-inflammatory immune

intracellularly or during sepsis responses
via TLR4 triggering PMNs to
release NETs.
• Shedding of platelet microparticles:
• Pathogen growth inhibition
- Formed via GPVI during RA, via
Via encapsulation and TLR4 during sepsis, or FcγRII by
secretion of antimicrobial bacterial/viral immune complexes
peptides, which may promote
formation of NETs - Microparticle cargo: transfer of
cytokines, chemokines,
• Pathogen elimination mitochondria, transcription factors,
lipids, enzymes, receptors, RNA,
- Possibly killing of the malarial
to various target cells
parasite P. falciparum inside red
blood cells
• MHC-class I:
- Redirecting the course of
Listeria monocytogenes from - Denatured on plasma membrane:
less immunogenic phagocytes immune suppression of CD8+ T
towards more immunologically cells
active splenic CD8α+ dendritic
- Intracellularly expressed upon
cells
platelet activation: activation of
antigen-specific CD8+ T cells
Fig. 17.1 Immune functions of platelets. The immune-sensing functions of platelets, generally
depicted as pathogen targeting and target cell communication
Conclusions
Platelets have traditionally been acknowledged as master regulators of hemosta-
sis. It has now, however, become evident that platelets are in fact much more
diverse and are capable of immune-sensing functions as well. Platelets can not
only enforce sophisticated protection mechanisms against invading pathogens
but are also capable of impacting and regulating immune functions in a large
variety of cells. They do so by utilizing numerous mechanisms, via diverse sur-
face molecules, through secretion of several pro- and inflammatory mediators
and through release of microparticles carrying a varying cargo. These relatively
novel aspects have shed new light on platelet functions beyond hemostasis and
will open up new avenues of research.
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Thrombopoietin Receptor Agonists:
Characteristics, Adverse Effects, 18
and Indications
Jenny Despotovic and Amanda Grimes
18.1 Introduction
While the most commonly utilized first-line therapeutics for the treatment of
immune thrombocytopenia (ITP) target the peripheral destruction of platelets, a pre-
ponderant second-line therapeutic agent targeting increased megakaryopoiesis and/
or platelet production has now emerged, in the form of thrombopoietin receptor
agonists (TPO-RAs). These TPO-RAs, eltrombopag and romiplostim, have been
proven safe and effective in treating ITP in both children and adults and have also
been approved for use in severe aplastic anemia and chronic hepatitis, with ongoing
investigation in the treatment of other clinical entities as well, including myelodys-
plastic syndrome and acute myelogenous leukemia, chemotherapy-induced throm-
bocytopenia, congenital thrombocytopenia, and other diseases. This chapter will
detail TPO-RA use in all of these different clinical settings.
18.2 Background
Thrombopoietin (TPO) is the major regulator of platelet production, acting largely

via the JAK and STAT signaling pathways to stimulate megakaryocyte growth and
platelet production (Kuter 2014). Shortly after identification and purification of
TPO by five different groups in 1994 (de Sauvage et al. 1994; Lok et al. 1994;
Bartley et al. 1994; Kuter et al. 1994; Kato et al. 1995), recombinant human TPO
products were created and tested in immune thrombocytopenia (ITP) and other
thrombocytopenic conditions. However, one of these agents resulted in the devel-
opment of antibodies to the recombinant TPO protein, causing neutralization of
J. Despotovic, DO, MS • A. Grimes, MD (*)

Pediatric Hematology/Oncology, Baylor College of Medicine/Texas Children’s Hospital,
6701 Fannin Street, Houston, TX 77030, USA

262 J. Despotovic and A. Grimes
both the recombinant TPO and endogenous TPO in some subjects, which resulted
in severe thrombocytopenia (Li et al. 2001). This resulted in cessation of further
development of recombinant TPO products. Development of less immunogenic
second-generation TPO receptor agonists (RAs) ensued. These second-generation
TPO mimetics bind to the TPO receptor at different sites, but both stimulate
increased platelet production via the same mechanism as endogenous TPO. There
are currently two TPO-RAs licensed for the treatment of ITP. Eltrombopag
olamine is a non-peptide small-molecule TPO mimetic which activates the TPO
receptor by binding to a transmembrane site on the receptor; and romiplostim is a
peptide-antibody (Fc fragment) fusion protein which activates the TPO receptor
by binding to its extra-cytoplasmic domain just as endogenous TPO does (Kuter
2014) (See Fig. 18.1) (Imbach and Crowther 2011). Both TPO-RAs have good
efficacy and favorable safety profiles and were approved by the US Food and
Drug Administration (FDA) for the treatment of chronic ITP in adults in 2008
(fda.gov 2017a, b). Eltrombopag received FDA approval for children with ITP in
2015 (fda.gov 2017a). These agents are common second-line treatment options
for ITP and now have expanding roles in other thrombocytopenic conditions
(Rodeghiero and Carli 2017).
18.3 Clinical Use and Pharmaceutical Considerations
Eltrombopag is approved for clinical use in children and adults with chronic ITP
refractory to or recurrent after first-line therapies (including intravenous immuno-
globulin [IVIG], anti-RhD immune globulin, glucocorticoids), adults with severe
aplastic anemia (SAA) refractory to first-line therapies (immunosuppressive ther-
apy) and ineligible for hematopoietic stem cell transplant (HSCT), and adults with
chronic hepatitis secondary to hepatitis C virus (HCV) infection (to enable inter-
feron therapy). Romiplostim is approved for clinical use in adults with chronic ITP
refractory to or recurrent after first-line therapies.
Eltrombopag is an oral medication that is taken once daily. It should be adminis-
tered on an empty stomach in order to maximize absorption. Additionally, eltrom-
bopag binds strongly to divalent cations and cannot be taken in close proximity to a
calcium-rich meal or the administration of multivitamin and/or mineral supple-
ments (2 h before or 4 h after), as this cation binding would prevent absorption of
the eltrombopag molecule. Dose should be titrated for a goal platelet count of
50,000/μL–200,000/μL, with desired platelet response typically achieved within an
average of 7 weeks (Neunert et al. 2016). Eltrombopag is generally well tolerated
and safe for long-term use. Romiplostim is administered via subcutaneous injection
once weekly. Dosage is titrated for a goal platelet count of 50,000/μL–200,000/μL,
with desired platelet response typically achieved within an average of 6 weeks
(Neunert et al. 2016). Generally, romiplostim therapy is also well tolerated and safe
for long-term use.
18 TPO-RAs: Characteristics, Adverse Effects, and Indications 263
a b
Fc domain Thrombopoietin
receptor binding
domains CO2H
OH
G NH
N
S S S S G
S S
N-terminal C-terminal H3C O
S S
S S S S G
N N
G
CH3
Interchain Intrachain Glycine
disulfide bonds disulfide bonds linker domains CH3
Romiplostim Eltrombopag
c
Thrombopoietin Romiplostim
receptor
Activation of
Extracellular thrombopoietin
receptor Eltrombopag
Cell membrane
of megakaryocyte
SHC
P JAK JAK P GRB2 RAS
SOS RAF
STAT
P
P
STAT
MAPK
Cytoplasm
Signal transduction P42 or P44
Increased platelet production
Fig. 18.1 Structure of romiplostim and eltrombopag and the cellular mechanisms of action. Panel
(a) shows the chemical structure of romiplostim, which is composed of the Fc portion of IgG1, to
which two thrombopoietin peptides consisting of 14 amino acids are coupled through glycine
bridges at the C-terminal of each γ heavy chain. Panel (b) shows the chemical structure of eltrom-
bopag. Panel (c) shows the cellular mechanisms of action of romiplostim, which binds to the
thrombopoietin receptor, and of eltrombopag, which binds to the thrombopoietin receptor’s trans-
membrane domain, thereby activating signaling that leads to increased platelet production. GRB2
denotes growth factor receptor-binding protein 2, JAK Janus kinase, MAPK mitogen-activated
protein kinase, P phosphorylation, RAF rapidly accelerated fibrosarcoma kinase, RAS rat sarcoma
GTPase, SHC Src homology collagen protein, and STAT signal transducer and activator of tran-
scription. From The New England Journal of Medicine, Paul Imbach and Mark Crowther,
Thrombopoietin-Receptor Agonists for Primary Immune Thrombocytopenia, Volume 365, Pages
734–741, Copyright © (2011) Massachusetts Medical Society. Reprinted with permission
18.4 A
dverse Effects and Theoretical Risks Associated
with TPO-RA Use
Overall, TPO-RAs are very well tolerated; and clinical data over the past 10–15 years
demonstrate that many of the theoretical risks potentially associated with TPO-RAs
have not been seen in clinical practice. However, some adverse effects of TPO-RAs
have been identified, and some theoretical risks require further investigation before
definitive conclusions regarding actual risk associated with TPO-RA use can be
made. Providers utilizing TPO-RAs in clinical practice should therefore be aware of
these risks and adverse effects. Potential risks associated with TPO-RA use include
thrombotic and/or thromboembolic complications, bone marrow fibrosis, rebound
thrombocytopenia, cataracts, hepatic abnormalities, development of cross-reactive
antibodies, and cytogenetic abnormalities/clonal evolution.
18.4.1 Adverse Effects and Risks Associated with both TPO-RA

Agents (Eltrombopag and Romiplostim)
18.4.1.1 Risk of Thrombosis

Increased risk of thrombotic complications in patients receiving TPO-RA therapy
has been a significant and closely monitored concern, with most studies confirming
a slightly increased risk of thrombosis among ITP patients receiving both eltrom-
bopag and romiplostim. However, the significance of this risk has remained uncer-
tain, given the potential biases within these studies, the preexisting increased
thrombotic risk among the ITP population, and the contribution of prior therapies
(i.e., splenectomy) to thrombotic risk. A recent review of industry-sponsored
eltrombopag and romiplostim investigations (Rodeghiero 2016) estimated the rate
of thromboembolic events to be 2.5–3.2 per 100 patient-years with eltrombopag and
4.2–7.5 per 100 patient-years with romiplostim. The rate of thromboembolic events
in ITP patients treated with TPO-RAs therefore appears to be higher than that in the
general population, as well as that in ITP patients not receiving TPO-RA therapy, in
which there is already ~two-fold increased risk of thromboembolism at baseline,
compared to the general population (Rodeghiero 2016). Notably, occurrence of
thromboembolic events was associated with advanced age and comorbid risk fac-
tors (hypertension, obesity, smoking, etc.), with the majority of data reported in
adult patients. Pediatric ITP patients treated with both romiplostim (Tarantino et al.
2016; Bussel et al. 2015a) and eltrombopag (Bussel et al. 2015b) reported no occur-
rence of thromboembolic events in initial industry trials, although two thromboem-
bolic events in pediatric ITP patients treated at ITP Consortium of North America
(ICON) sites (2.5%), both receiving eltrombopag, were later reported (Neunert
et al. 2016), with overall incidence of thrombosis among pediatric ITP patients
receiving TPO-RA therapy remaining very low. Thrombotic risk in ITP patients
treated with TPO-RAs does not appear to correlate linearly with the platelet count
and also does not appear to correlate consistently with medication dose (Rodeghiero
et al. 2013; Saleh et al. 2013). The extent of thrombotic risk associated with TPO-RA
therapy is still being established, with ongoing investigation needed to verify true
incidence rates and/or associations while controlling for confounding variables. As
these data are further clarified, providers should be aware of this risk in clinical
practice and evaluate individual risk factors on a case-by-case basis when consider-
ing initiation of TPO-RA therapy. Additionally, dosage should be titrated to achieve
and maintain the minimum platelet count needed to reduce the risk of bleeding
(≥50,000/μL), but attempt should not be made to normalize platelet count. Care
should be taken to avoid thrombocytosis, as this could potentially contribute to the
risk of thrombosis.
18.4.1.2 Risk of Bone Marrow Fibrosis

Accelerated bone marrow fibrosis is another theoretical risk associated with TPO-RA
therapy. Given that TPO-RAs work by stimulating megakaryopoiesis and increased
platelet production, it is thought that profibrotic cytokines expressed by megakaryo-
cytes and platelets will also be increased via the action of TPO-RAs on the TPO
receptor (Kuter et al. 2007). Two large studies evaluating chronic ITP patients receiv-
ing long-term TPO-RA therapy showed a low incidence of bone marrow fibro-
sis—1.7% in patients receiving eltrombopag for up to 5.5 years (Brynes et al. 2015)
and 2% in patients receiving romiplostim for up to 5 years (Rodeghiero et al. 2013).
In these large adult trials, the majority of bone marrow fibrosis was restricted to
reticulin fibrosis, with rarely identified collagen fibrosis (Rodeghiero et al. 2013;
Saleh et al. 2013; Brynes et al. 2015). Reticulin fibers, best seen with a reticulin stain,
can be seen normally in the bone marrow of healthy individuals, although increased
levels of reticulin fibrosis may be associated with various disease processes and, in
fact, have been demonstrated in adult ITP patients who are treatment naïve (present
in 31% of ITP patients yet to receive therapy in one study) (Rizvi et al. 2015).
Collagen fibrosis, conversely, is not present in healthy bone marrow and always
reflects an underlying disease process—most often a myeloproliferative disorder.
These fibers are composed of type I collagen and are best seen with a trichrome stain.
The one ITP patient receiving romiplostim therapy in whom collagen fibrosis was
identified also had known preexisting cytogenetic abnormalities associated with
myelodysplastic syndrome (Rodeghiero et al. 2013). Also of note, identification of
bone marrow fibrosis in chronic ITP patients receiving eltrombopag or romiplostim
did not correlate with peripheral blood count abnormalities or other unexpected mor-
phological or quantitative bone marrow abnormalities and also appeared to be revers-
ible or stable once TPO-RA therapy was discontinued (Rodeghiero et al. 2013; Saleh
et al. 2013; Brynes et al. 2015). Although smaller studies have shown more inconclu-
sive results, the larger body of evidence regarding accelerated bone marrow fibrosis
associated with TPO-RA therapy to date demonstrates no significant concern for this
risk. Therefore, experts generally do not recommend routine bone marrow monitor-
ing in patients receiving TPO-RA therapy, but continued monitoring of peripheral
blood counts and smear should occur on a routine basis; and if cytopenias or morpho-
logical abnormalities develop, a bone marrow evaluation should be obtained.
18.4.1.3 Risk of Rebound Thrombocytopenia

Rebound thrombocytopenia following discontinuation of TPO-RA therapy is a well-
documented risk, and many hematologists wean rather than abruptly discontinue
these agents, with continued close follow-up for at least 2–4 weeks following discon-
tinuation of therapy. The majority of ITP patients receiving TPO-RA therapy return
to pre-therapy baseline platelet levels upon discontinuation of therapy, with a small
subset of patients actually achieving sustained platelet responses following discon-
tinuation of therapy (Ghadaki et al. 2013; Bussel et al. 2015c; Biagiotti et al. 2015).
Up to 10% of patients experience significant rebound thrombocytopenia, with plate-
let counts decreasing below pretreatment levels and remaining low for 1–3 weeks
before returning to prior baseline (Kuter et al. 2008). This is likely due to the fact that
endogenous TPO activity is regulated by platelet mass, which may be suppressed
while platelet levels are elevated on TPO-RA therapy and cannot quickly equilibrate
when therapy is abruptly discontinued. This rebound thrombocytopenia can be asso-
ciated with significant risk of bleeding. Therefore, although manufacturers of both
agents recommend holding the dose if the platelet count exceeds 400,000/μL, expe-
rienced providers recommend close monitoring and dose reduction, as well as initia-
tion of low-dose ASA if platelet count exceeds 800,000/μL, to avoid the risk of
rebound thrombocytopenia, which in clinical practice likely poses higher risk to the
patients than a transiently elevated platelet count (Kuter et al. 2010).
18.4.1.4 Risk of Clonal Evolution

Given prior knowledge of hematopoietic precursor cell expression of c-mpl (TPO
receptor), the direct action of TPO on the hematopoietic stem cells was established
shortly after its purification and successful cloning in 1994 (Zeigler et al. 1994). This
direct stimulation of early hematopoietic stem cells by TPO is the basis for investigat-
ing the use of TPO-RAs in refractory and, now frontline, severe aplastic anemia (SAA).
However, this is also the basis for the theoretical concern about clonal evolution with
the use of TPO-RAs in both SAA and myelodysplastic syndromes (MDS)/acute
myelogenous leukemia (AML). Recent studies evaluating the effects of eltrombopag
therapy in SAA have shown new cytogenetic abnormalities/clonal evolution in 8–18%
of SAA patients treated with eltrombopag (Desmond et al. 2014; Townsley et al. 2015;
Gill et al. 2017; Townsley et al. 2017), which appears consistent with the incidence of
clonal evolution in historical cohorts of SAA patients treated with immunosuppressive
therapy alone. This likely represents a risk associated with the disease and not eltrom-
bopag therapy, but further investigation is needed to clarify the potential risks of clonal
evolution in the treatment of SAA and MDS/AML with TPO-RAs.
18.4.2 Adverse Effects and Risks Associated with Eltrombopag
18.4.2.1 Risk of Cataract Formation

There was initial concern for increased risk of cataract formation related to eltrom-
bopag therapy, due to preliminary findings in animal studies. However, no clinical
investigation has shown an increased risk of cataracts with eltrombopag use (Bussel
et al. 2009; Cheng et al. 2011). There have been cataracts reported in patients treated
with both eltrombopag and romiplostim, but these patients were also exposed to
significant steroid doses. There does not appear to be an increased risk of cataract in
children. Some experts continue to recommend baseline ophthalmologic evaluation
prior to initiation of eltrombopag therapy and annually while continuing therapy, as
further clinical data accumulates.
18.4.2.2 Risk of Liver Function Abnormalities

Hepatic abnormalities have been noted with eltrombopag therapy. Elevations in
serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bili-
rubin occurred in 4–6% of patients receiving eltrombopag therapy (Novartis 2016).
Although these hepatobiliary laboratory abnormalities are generally asymptomatic
and appear to resolve with disruption or discontinuation of therapy, certain monitor-
ing parameters and corresponding dose titration or discontinuation actions are rec-
ommended to prevent drug-induced liver injury. Large trials reporting the safety of
eltrombopag therapy in ITP patients generally recommend discontinuing drug when
the following parameters are met: ALT ≥ 5× the upper limit of normal and/or
ALT ≥ 3× the upper limit of normal with accompanying hepatitis symptoms or rash
and bilirubin ≥1.5× the upper limit of normal (with >35% direct fraction) (Saleh
et al. 2013). Additionally, providers utilizing eltrombopag in the setting of thrombo-
cytopenia related to chronic HCV-associated hepatitis, specifically in combination
with ribavirin- and interferon-based therapies, need to be aware of the increased risk
for acute hepatic decompensation in this setting (fda.gov 2017a).
18.4.2.3 Other Adverse Effects Associated with Eltrombopag

Milder side effects noted most commonly in patients receiving eltrombopag therapy
include headache, nasopharyngitis, upper respiratory tract infection, and fatigue
(Saleh et al. 2013). However, eltrombopag therapy is generally well tolerated.
18.4.3 Adverse Effects and Risks Associated with Romiplostim
18.4.3.1 Risk of Antibody Development

Previous experience with the first-generation recombinant TPO proteins and resul-
tant development of cross-reactive antibodies with the use of the pegylated recom-
binant product generated persistent concern for the development of similar
antibodies with the use of second-generation TPO-RAs as well. Eltrombopag car-
ries no relevant risk for cross-reactive or neutralizing antibody formation though, as
it is a small-molecule therapeutic and also shares no structural or epitope homology
with endogenous TPO; and in clinical practice, no antibody formation has been
noted. Conversely, romiplostim does carry a theoretical risk of cross-reactive anti-
body formation, as it acts on the same TPO receptor extra-cytoplasmic binding
domain as endogenous TPO acts upon, albeit without any structural similarity to
endogenous TPO. However, no cross-reactive antibody formation has occurred in
10–15 years of clinical romiplostim use. Romiplostim therapy does result in
formation of neutralizing anti-romiplostim antibodies in a small subset of patients,

which results in a decrease or cessation of treatment response. This phenomenon
should therefore be considered and further investigated in patients previously
responsive to romiplostim therapy, with subsequent loss of response (Neunert et al.
2016; Rodeghiero et al. 2013; Carpenedo et al. 2016).
18.4.3.2 Other Adverse Effects Associated with Romiplostim

The most commonly noted side effects in patients receiving romiplostim therapy are
headache, fatigue, and nasopharyngitis (Rodeghiero et al. 2013), although as previ-
ously noted, romiplostim is well tolerated overall.
18.5 U
se of Thrombopoietin Receptor Agonists in
Immune Thrombocytopenia (ITP)
TPO-RA therapy is most extensively studied and widely utilized in the treatment of
patients with ITP. Early clinical trials investigating the use of both eltrombopag and
romiplostim in adults with chronic ITP resulted in an FDA indication for both
agents in adult patients with chronic ITP refractory to first-line therapies (IVIG,
anti-D immune globulin, and glucocorticoids) (fda.gov 2017a, b). Eltrombopag and
romiplostim response rates in treatment-refractory chronic ITP patients, generally
defined by achievement of platelet count ≥50,000/μL, averaged ~80% (compared to
an average of 20% or less in placebo groups), also with decreased bleeding events,
improved health-related quality of life scores, and no increased incidence of severe
adverse effects noted in eltrombopag (Saleh et al. 2013; Bussel et al. 2009; Cheng
et al. 2011; Bussel et al. 2007; Bussel et al. 2013) and romiplostim (Rodeghiero
et al. 2013; Kuter et al. 2008; Kuter et al. 2010) treatment groups. Additionally,
follow-up studies demonstrated maintenance of sustained treatment response and
continued safety in chronic ITP patients receiving long-term eltrombopag (Saleh
et al. 2013) or romiplostim (Rodeghiero et al. 2013) therapy for up to 3–5 years.
Similarly, studies in pediatric ITP patients (≥1 year of age) have demonstrated the
safety and efficacy of eltrombopag in this population (Bussel et al. 2013; Grainger
et al. 2015), with an FDA indication for the use of eltrombopag in pediatric chronic
ITP patients refractory to first-line therapies. Likewise, ongoing studies show excel-
lent safety and efficacy profiles for romiplostim use in the treatment of pediatric ITP
(Tarantino et al. 2016; Bussel et al. 2011), and this therapy is also widely utilized
clinically for the treatment of childhood ITP.
In clinical practice, the role of TPO-RA therapy in ITP continues to expand signifi-
cantly as further data proving the safety and efficacy of these agents is compiled. For
example, TPO-RA therapy is occasionally implemented earlier in the treatment of
symptomatic ITP, in an attempt to avoid splenectomy or extensive glucocorticoid
exposure, prior to possible disease resolution over the first 12 months. These agents
may be easily weaned during the persistent phase, as spontaneous recovery occurs.
Alternatively, therapy may be continued or reinitiated as indicated, if chronic disease
develops. Studies have shown that response is maintained with intermittent use
(i.e., response is again achieved at similar levels when TPO-RA therapy is resumed)

and extended use (Rodeghiero et al. 2013; Bussel et al. 2013) and that therapy can be
discontinued with no irreversible or long-term complications once ITP resolves
(Ghadaki et al. 2013). Moreover, studies show that up to 30% of ITP patients treated
with TPO-RAs maintain an extended response (up to 6 months and longer) once ther-
apy is discontinued (Ghadaki et al. 2013; Bussel et al. 2015c; Biagiotti et al. 2015),
with the implication that TPO-RA therapy may be capable of inducing a sustained
platelet response. More long-term follow-up data is needed to define this possibility.
The advent of TPO-RA therapies has broadened the therapeutic landscape in
ITP. TPO-RAs induce reliable platelet responses in the majority of patients and are
also associated with decreased bleeding events, improved health-related quality of
life, and an overall favorable safety profile, including the lack of immunosuppression
associated with other second-line therapies. When one TPO-RA is ineffective or not
tolerated, response or tolerability could be achieved by switching to an alternate
TPO-RA, with no cross-resistance noted between the two available drugs in this class.
In summary, abundant data supports consideration of treatment with TPO-RA
therapies in both adults and children with chronic ITP. Treated patients often have
reduced bleeding complications, fewer activity restrictions, and potentially
improved quality of life (Tarantino et al. 2016; Bussel et al. 2013; Rodeghiero et al.
2013; Saleh et al. 2013; Kuter et al. 2008; Kuter et al. 2010; Bussel et al. 2009;
Cheng et al. 2011; Bussel et al. 2007; Bussel et al. 2013; Grainger et al. 2015;
Bussel et al. 2011). Additionally, patients may be able to avoid splenectomies or
adverse effects of prolonged immunosuppression. Occasional sustained platelet
responses have occurred after TPO-RA therapy is discontinued, but these agents
should not be considered curative.
18.6 U
se of Thrombopoietin Receptor Agonists in
Severe Aplastic Anemia (SAA)
Outside of therapy for chronic ITP, eltrombopag has a clinical indication for the
treatment of patients with SAA who are refractory to first-line immunosuppressive
therapies and are ineligible for hematopoietic stem cell transplant (HSCT). In its
original use, eltrombopag was intended to ameliorate the complications of thrombo-
cytopenia which are associated with SAA. However, during initial trials, trilineage
effects of eltrombopag were noted, with improved red blood cell (RBC) and white
blood cell (WBC) parameters in addition to the improved megakaryopoiesis and
platelet production which was expected (Desmond et al. 2014). As discussed previ-
ously, this is likely due to the direct action of the TPO-RAs on early hematopoietic
precursors via the same mechanism as endogenous TPO (Zeigler et al. 1994), creat-
ing expansion of all hematopoietic stem cells and thereby all cell lines. This finding
prompted further investigation of eltrombopag as therapy for SAA, with ongoing
trials now investigating this medication as a frontline therapeutic option (in combina-
tion with immunosuppressive therapy) for SAA. Early results are promising, with a
recently published trial enrolling 92 SAA patients to receive eltrombopag in
combination with immunosuppressive therapy as frontline therapy (starting at either

Day 1 or Day 14) showing average complete response rates of 39% at 6 months and
overall response rates of 87% at 6 months, compared to complete response rates of
10% and overall response rates of 66% in historical SAA cohorts treated with immu-
nosuppressive therapy alone (Townsley et al. 2017). Incidentally, greatest response
rates were noted in the patient cohort beginning eltrombopag therapy at Day 1. In all
patients receiving frontline eltrombopag, marked increases in bone marrow cellular-
ity, CD34+ stem cells, and early hematopoietic progenitors were noted. Hematopoietic
progenitor stimulation does raise the concern for potential development of new cyto-
genetic abnormalities or clonal evolution in SAA, given that up to 1/3 of AA/SAA
patients have genetic mutations typically associated with myeloid neoplasms
(Yoshizato et al. 2015). However, rates of clonal evolution in this study of 92 SAA
patients treated with frontline eltrombopag therapy were similar to those in historical
cohorts treated with immunosuppressive therapy alone (~8%) (Townsley et al. 2017).
Further investigation of TPO-RAs in the treatment of SAA is needed at this point;
however, preliminary findings portend a possible paradigm shift in the management
of SAA, with the introduction of eltrombopag as a frontline treatment option.
18.7 U
se of Thrombopoietin Receptor Agonists in Chronic
Hepatitis
A third clinical indication for the use of eltrombopag is in the treatment of chronic hepa-
titis C virus (HCV)-associated hepatitis. The premise of this indication is based on the
frequent need for peginterferon-based therapy in patients with chronic HCV infection,
which cannot be effectively completed in the setting of comorbid thrombocytopenia.
Eltrombopag has proven effective in raising platelet counts in this population, leading to
decreased bleeding complications and, most importantly, to successful completion of
peginterferon therapy without interruptions, dose reductions, or discontinuations. This
has led to an increased rate of sustained virological response during peginterferon-based
antiviral therapy in this population of patients (Fried et al. 2002). As peginterferon ther-
apy becomes less utilized in the advent of newer antiviral therapies for HCV infection,
this indication for eltrombopag therapy may change. However, both eltrombopag and
peginterferon therapy will likely continue to play a significant role in HCV-associated
hepatitis therapy for an extended period of time, until these newer antiviral agents
become more widely available and accessible throughout the world.
18.8 O
ff-Label Uses of Thrombopoietin Receptor Agonists,
Currently Under Investigation
18.8.1 First-Line Treatment for Aplastic Anemia
Investigation of eltrombopag as first-line treatment in SAA is discussed in detail

above (Use of TPO-RAs in SAA). Early results are promising, likely owing to
generalized stimulation of early hematopoietic precursor cells by eltrombopag, ulti-

mately resulting in multi-lineage effects.
18.8.2 Myelodysplastic Syndrome (MDS)/Acute

Myelogenous Leukemia (AML)
The premise for investigation of TPO-RAs in the treatment of MDS and AML is
similar to that in SAA. Thrombocytopenia is a major problem in these patients,
occurring in 40–65% (Kantarjian et al. 2007) and contributing significantly to
bleeding complications and morbidity/mortality in this population of patients.
Eltrombopag and romiplostim are expected to ameliorate the thrombocytopenic
complications associated with MDS and AML if found to be safe and effective.
There are currently many ongoing trials among pediatric and adult patients with
MDS/AML, mostly in advanced or refractory patients (with eltrombopag) but also
in lower-risk patients (with romiplostim). Generally, treatment with both TPO-RAs
has been demonstrated to be safe, although there was early concern for increased
progression to AML in patients treated with romiplostim (with updated findings less
clear that there is any increased risk of AML progression associated with romiplos-
tim therapy (Kantarjian et al. 2015)).
Eltrombopag has been studied in 98 relapsed or refractory adult patients with
intermediate-2- or high-risk MDS or AML, with good results. Rates of platelet and
red blood cell transfusion independence were improved in the eltrombopag-treated
patients vs patients receiving placebo therapy (38% vs 21% and 20% vs 6%, respec-
tively) (Platzbecker et al. 2015). In another trial investigating the efficacy and safety
of eltrombopag in intermediate-2- or high-risk MDS or AML patients, the frequency
of clinically relevant thrombocytopenic events and severe bleeding was also
decreased in the eltrombopag treatment group (Mittelman et al. 2016). In both of
these trials, there was no increased frequency of disease progression in the
eltrombopag-treated patients; and in fact, in the latter trial (Mittelman et al. 2016),
there was a reduced trend of disease progression in the eltrombopag-treated arm vs
placebo (42% vs 60%, respectively). Another ongoing study investigating eltrom-
bopag therapy in adult patients with relapsed or refractory low- and intermediate-1-
risk MDS has shown promising results as well, with 54% of patients receiving
eltrombopag therapy having achieved a platelet response at 24 weeks, compared to
27% of patients receiving placebo therapy. Again, the incidence of AML evolution
or MDS disease progression was not increased in eltrombopag-treated patients; and
of note, six patients treated with eltrombopag had gone into complete remission at
24 weeks (Oliva et al. 2015).
Romiplostim therapy in MDS has been studied largely in lower-risk patients. In
one year-long study in low- and intermediate-1-risk MDS patients receiving sup-
portive care only with platelet counts ≤50,000/μL, a durable platelet response was
achieved in 46% of romiplostim-treated patients. Notably, progression to AML was
observed in ~5% of patients (two patients) (Kantarjian et al. 2010a). Additional
smaller studies investigating the safety and efficacy of romiplostim in combination
with azacitidine (Kantarjian et al. 2010b), lenalidomide (Wang et al. 2012), and
decitabine (Greenberg et al. 2013) in lower-risk MDS patients all demonstrated
improved thrombocytopenic events and decreased bleeding events in romiplostim-
treated patients. Numbers of patients included in each study were too small to deter-
mine romiplostim effects on disease progression. At this time, further studies are
needed to clarify whether romiplostim therapy in MDS or AML poses a risk for
accelerated disease progression. Eltrombopag, however, appears not to be associ-
ated with risk for disease progression in MDS and AML patients (Platzbecker et al.
2015; Mittelman et al. 2016; Oliva et al. 2015). This may be due to eltrombopag’s
ancillary property as a divalent ion chelator—specifically as an iron chelator in this
instance (Roth et al. 2012). Again, further investigations regarding potential antitu-
mor properties of eltrombopag are yet to be completed in the clinical setting and
will need further exploration before definitive associations are made.
18.8.3 Chemotherapy-Induced Thrombocytopenia
TPO-RAs have not yet been extensively studied in the setting of chemotherapy-
induced or radiation-induced thrombocytopenia and/or bone marrow failure.
However, small studies investigating both eltrombopag and romiplostim use among
patients receiving chemotherapy for solid tumors have shown favorable results. For
both TPO-RA agents, treatment resulted in decreased platelet count nadirs and
fewer dose modifications and/or interruption of chemotherapy (Winer et al. 2015;
Kellum et al. 2010; Parameswaran et al. 2014). Larger randomized trials are needed
to determine safety and to better identify optimal treatment groups for TPO-RA
therapy in chemotherapy-induced thrombocytopenia prior to recommending
TPO-RA therapy in this clinical setting.
18.8.4 Thrombocytopenia Following Hematopoietic

Stem Cell Transplant (HSCT)
Delayed platelet recovery following HSCT is a common problem associated with

HSCT in both children and adults. No extensive investigation regarding the use of
TPO-RA therapy in the setting of post-HSCT thrombocytopenia has been done.
However, early and ongoing studies are promising, confirming both safety and effi-
cacy of TPO-RAs in this setting. An ongoing phase 2 randomized controlled trial
evaluating the safety and efficacy of eltrombopag in the treatment of delayed plate-
let recovery (≥35 days after HSCT) in adult patients undergoing HSCT demon-
strated platelet response (≥50,000/μL) in 21% of eltrombopag-treated patients at
8 weeks, vs 0% of patients receiving placebo. Additionally, there was no increased
morbidity/mortality or incidence of adverse effects in the group of patients receiv-
ing eltrombopag therapy (Popat et al. 2015). Similarly, two case series evaluating
the use of romiplostim in delayed platelet recovery post-HSCT showed no increased
adverse effects in romiplostim-treated patients and demonstrated realization of
platelet response (≥50,000/μL) in all patients treated within 2–12 weeks (Calmettes

et al. 2011; Poon et al. 2013). Further data is needed to determine safety and effi-
cacy of TPO-RA approach for management of thrombocytopenia following HSCT,
but early results are promising.
18.8.5 Congenital Thrombocytopenias
Very little investigation regarding the role of TPO-RAs in the setting of hereditary
thrombocytopenias has been completed. However, we know that this is a rapidly
expanding category of diseases given the ongoing advances in genetic diagnostic
tools, with potential utility of TPO-RA therapy. Small studies have shown some
benefit of eltrombopag therapy in both Wiskott-Aldrich syndrome and myosin
heavy chain 9 (MYH-9)-related thrombocytopenias; but more studies are needed to
delineate the safety and efficacy of TPO-RA therapy in this clinical setting.
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Registries in Immune
Thrombocytopenia: The History 19
of the Intercontinental Cooperative ITP
Study Group
Thomas Kühne
19.1 Introduction
Immune thrombocytopenia (ITP) is an acquired autoimmune-mediated thrombocy-

topenia, defined by a platelet count of <100 × 109/l based on the definitions of an
international working group (Rodeghiero et al. 2009). Besides bleeding, patients are
healthy, and except thrombocytopenia, all other hematological values including
blood smear analysis are normal. Thrombocytopenia is the result of an increased
destruction of autoantibody-coated platelets by the monocytic-phagocytic system
and a certain degree of platelet production failure, because megakaryocytes in the
bone marrow express the same epitopes as do platelets and are thus affected by
destructive processes, which may impair development and induce apoptosis of
megakaryocytes (Perera and Garrido 2017).
ITP was often referred to as Werlhof’s disease according to Paul Gottlieb
Werlhof, who published in 1735 a report of a female suggestive to be one of the first
descriptions of ITP (Werlhof 1735). However “Morbus” Werlhof implicates a dis-
ease with a unique etiology and pathomechanism and would ask therefore for a
standardized algorithm of the management. If pathophysiology could be identified
by routine laboratory tests in each patient, probably patients would be treated very
differently. Thus a unique approach to patients with ITP, as it is currently practiced,
appears to be questionable.
Autoantibodies are not found in every patient with ITP and patients with ITP do
not respond to standard therapy in the same way, suggesting that besides autoreac-
tive antibodies, there are different mechanisms ultimately resulting in thrombocyto-
penia, such as cytotoxic T lymphocyte-mediated cellular toxicity (Zufferey et al.
T. Kühne, MD
Division of Oncology/Hematology, University Children’s Hospital,
Postfach, Spitalstrasse 33, CH-4031 Basel, Switzerland

278 T. Kühne
2017). This mechanism has the potential to destroy platelets and to affect their
development by inducing megakaryocyte maturation defects (Stasi 2012). In con-
trast to secondary ITP, that is defined by a known underlying disorder or trigger,
primary ITP is of unknown etiology and represents a diagnosis of exclusion without
any specific laboratory tests, leaving the diagnosis of primary ITP vague and impre-
cise. Secondary ITP is much rarer than primary ITP.
Children and adolescents of all age groups but also adults and elderly patients
may exhibit age-specific clinical characteristics. The bleeding phenotype can be
affected by coexisting factors other than thrombocytopenia, i.e., exogenous factors
(e.g., infectious diseases, drugs), endogenous factors (e.g., age, arteriovenous mal-
formations, gene polymorphisms influencing immune response or hemostatic reac-
tions, and inherited disorders of hemostasis, such as von Willebrand disease or
inherited platelet function disorders), comorbidities, drugs, and other factors (Kühne
2017; Michel et al. 2011; Kistanguri and McCrae 2013).
In 1996 the American Society of Hematology (ASH) published a clinical prac-
tice guideline article with a comprehensive literature review of the time between
1966 and 1994 (George et al. 1996). It was recognized that the management of
children and adults with ITP is mainly based on opinion rather than on evidence-
based decisions. This publication stimulated a group of pediatric hematologists to
establish an international network of scientists and physicians involved in the field
of ITP with the aim to promote evidence-based medicine (Imbach et al. 2002;
Kühne 2013). The name of the group was Intercontinental Childhood ITP Study
Group (ICIS), which was changed in 2004 to Intercontinental Cooperative ITP
Study Group (ICIS), because of collaboration with adult hematology (www.itpba-
sel.ch). The research activities of ICIS include the planning, performing, and assess-
ing of projects with four international registries, two of them being still open for
patient registration, exchange and interpretation of data, and organization of regular
meetings. This review article summarizes the activities of ICIS and reviews regis-
tries in patients with ITP (Table 19.1).
Table 19.1 Intercontinental Cooperative ITP Study Group projectsa

Duration of Investigators Institutions Countries
Name of the project project Patients (N) (N) (N) (N)
Registry I 1997–2000 2786 228 153 41
Registry II 2002–2004 1383 96 69 42
Splenectomy Registry Since 1998 357 80 63 27
PARC-ITP Pediatric Since 2004 3995 107 92 34
and Adult Registry on
Chron. ITP
As of March 31, 2017
a
19 Registries in Immune Thrombocytopenia 279
19.2 Methods in ITP
The documentation and classification of quality of the management in medicine and

the development of strict definitions of evidence-based medicine by various sys-
tems, such as the GRADE system, are important achievements. These systems use
a systematic and validated approach to grading the strength of clinical practice rec-
ommendations to minimize the potential for bias, to promote evidence-based medi-
cine, and to enhance the validity of interpretations of research results (Guyatt et al.
2008). Different methods and strategies have been developed and practiced, in order
to qualify recommendations and medical decisions based on their background
(opinion or evidence).
Primary ITP with its complex pathophysiology and unknown etiology represents
a disturbance of the immunological balance between self and nonself. Because it is
not possible to investigate the exact pathomechanisms in the daily routine, the diag-
nosis is associated with imprecision and vagueness. Patients with primary ITP are
characterized sometimes with a change in their diagnosis to secondary ITP or to
thrombocytopenia of other reasons, if an underlying cause of thrombocytopenia
becomes evident. Therefore classification of ITP can be difficult and the disease
may manifest an evolutionary change of diagnostic features, which may ask for
adaptations in the management. The clinician caring for patients with ITP must
always consider the adequacy of the diagnosis and be ready to initiate new diagnos-
tic procedures, if new insights arise, such as changes in physical signs and symp-
toms, new laboratory results, therapy refractoriness, and other unexpected clinical
occurrences. Constant adaptations of the clinical management are typical for auto-
immune disorders. Practice guidelines and recommendations exhibit a certain
degree of rigidity in following such an evolution and do not replace best physical
judgments, making physicians’ experience and networking important prerequisites
to manage children and adults with ITP. General algorithms for the management of
patients with ITP must be therefore interpreted carefully, particularly in patients
with chronic ITP. The management of patients with ITP is characterized by a rather
individualized concept, which can be further influenced by patients’ preferences,
making the measurement and interpretation of health-related quality of life impor-
tant (Haverman et al. 2017).
Evidence-based medicine qualifies research according to strict rules. For exam-
ple, the revised ASH 2011 evidence-based practice guideline used the GRADE sys-
tem, which categorizes the quality of the contributing evidence and the strength that
the evidence brings to the recommendations (Neunert et al. 2011). The system con-
sists of numbers indicating the strength of a recommendation and letters indicating
the quality of the underlying evidence, e.g., randomized controlled trials, observa-
tional studies, or case series. Additionally, the process of achieving a recommenda-
tion must include a declaration of all individuals’ potential and existing conflicts of
interests, which may influence such recommendations.
280 T. Kühne
Registries are widely used in medicine and represent ideal scientific tools for the
study of rare diseases with the opportunity to collect multicenter data of a large
number of patients from all continents within short time period (Gliklich et al.
2014). Registry data may be the basis to elaborate scientific questions and to prepare
clinical trials, particularly to investigate inclusion and exclusion criteria of patients,
to evaluate the relevance of scientific objectives and the feasibility of clinical
research. The design and infrastructure of the registry depend upon the require-
ments and research plans of the research groups, the epidemiology of the disease to
be investigated, and the availability and duration of funding (Gliklich et al. 2014).
There are attractive features and advantages of registries that include an imme-
diate access to research, interim analyses, and a structure that allows flexibility
with adaptations and modifications when new scientific questions arise during
data analyses or new knowledge occurs from the scientific community. Additionally
a registry permits “learning effects” during data acquisition, resulting in consecu-
tive changes of registry questions and structures. The database is often accessible
by the Internet and allows fast reporting and structured screening of entered data
by the system but also by the central office as part of a quality program to avoid
erroneous data.
There are also limitations of registries. Despite its flexibility, registries are under
control of scientific groups and regular authorities, and thus changes of their struc-
ture could be associated with high work load and need adaptations by their users.
There are both population-based national and international registries and case-based
registries. The former type presumes a controlled and thus costly endeavor to
include a preferably complete number of patients representing the population of a
given geographical area. Often, such registries are designed for other purposes than
the study of a specific disease or for the study of a disease group, such as public
health and cancer registries. Exact case definitions are often missing in such regis-
tries. Case-based registries depend often on voluntary registration by physicians
participating in a network. They are often designed for the study of a specific dis-
ease and benefit from its flexibility by changes of its structure, should new data and
knowledge of the disease occur. There are also economic problems particularly for
investigator-driven case-based registries. Remuneration of participating investiga-
tors and institutions and financial aspects of the development and maintenance of
the database and its structures must be considered for budgets. Databases of regis-
tries depend on sophisticated software applications and need specialists in informat-
ics. International multicenter data may contain multilevel biases based on country-,
region-, and institution-specific characteristics which may affect quality. However,
quality can be controlled by the structure of the registry, by continuous modifica-
tions of Internet access software, by the knowledge of participating institutions and
investigators, and by quality control measures, such as regular interim analyses and
on-site monitoring procedures. Quality control can be expensive and must be con-
sidered in preparing budgets. Case-based registries collect data of consecutive
patients by voluntary reporting of investigators. This must be considered when
assessing results.
19.3 History and Development of ICIS
In 1997 ICIS was founded and started immediately with the activation of registries.
Based on the lack of clinical data in ITP and the fact that many, if not most clinical
decisions in children with ITP were based on expert opinion (George et al. 1996),
the objectives of ICIS included the establishment of a well-functioning and acces-
sible international network of scientists and clinicians involved in the field of throm-
bocytopenia and ITP, the prospective collection of clinical and laboratory data from
children with ITP by registries, and regular publications and organization of meet-
ings to exchange experience and scientific achievements. In 2004 ICIS decided to
change its structure to a group of pediatric and adult hematologists, because ITP—
although different in some aspects—shares many common characteristics through-
out age groups, including the rarity, the challenges to perform research, and clinical
features. Furthermore the extension of the group will benefit from an increased
strength of more experts in the field. The ICIS registries represent ideal tools to
learn more about demographics of patients, the natural history of ITP, and manage-
ment issues.
Since 2003, ICIS performs international expert meetings every third year in
Switzerland and published them in peer-reviewed scientific journals (Imbach et al.
2003; Kühne and Imbach 2006, 2010, 2013, 2016). These meetings are opportuni-
ties for experts to exchange knowledge and to discuss further projects. ICIS investi-
gators can share their research results and get most actual information in these
meetings. The most recent meeting took place in 2015 in central Switzerland with
the topic “immunomodulation and management in ITP and other autoimmune dis-
orders.” For the first time, experts not only from hematology and basic sciences but
also from other disciplines with autoimmune disorders, such as dermatology, rheu-
matology, pneumology, immunology, neurology, and gastroenterology, were invited
to give a lecture and write a manuscript on the topic “immunomodulation.” The
meeting was an exciting and successful scientific exchange of many experts (Kühne
and Imbach 2016). ICIS is currently preparing its sixth expert meeting, which will
be held in Arosa, Switzerland, in 2018 with the topic “to treat or not to treat.”
Another stimulating scientific event can be expected.
19.4 ICIS Registry I (1997–2000)
Registry I was the first initiative by ICIS with the aim to collect data of children with
ITP from all continents within short time period and to learn more about the natural
history of pediatric ITP in different countries. It was designed to register data at the
time of first presentation, with follow-up data 6 and 12 months later. The manage-
ment and outcome was assessed with a main focus on watchful waiting, corticoste-
roids, and intravenous high-dose immunoglobulins (IVIG). Findings of several
analyses included:
282 T. Kühne
( 1) Clinical characterization of children of different age groups.

(2) The percentage of children with chronic ITP at 6 and 12 months (at that time
chronic ITP was defined with 6 months’ duration of thrombocytopenia, and
thrombocytopenia was defined with a platelet count of <150 × 109/l): 31% had
chronic ITP without a difference between boys and girls.
(3) A low frequency of severe and life-threatening bleeding despite a very low
platelet count of less than 20 × 109/l at presentation. Intracranial hemorrhage
was reported in 2 of 1496 children with data available at 6 months after the
initial presentation.
(4) Predictive factors for chronic ITP included a higher platelet count at diagnosis
and older age.
(5) The outcome of children at 6 months after initial presentation did not depend on
whether they received drug treatment or were managed without drugs (Kühne
et al. 2001).
Another analysis revealed age-specific characteristics of ITP, including 203

infants who exhibited a lower incidence of chronic ITP (Kühne et al. 2003). It has
been shown that boys are more frequently affected with ITP during infancy. The
reason for this observation remains unclear. Interestingly it has been shown that
among older adults men are again more frequently seen than women (Moulis et al.
2014). An important observation was that 25% of children recovered from their ITP,
who were observed during 6 and 12 months after the diagnosis of ITP (Imbach et al.
2006). The maintained potential of remission during 1 year resulted in a discussion
at an ICIS expert meeting and finally leads to a change of the definition of chronic
ITP to be of rather 12 than of the earlier used 6-month duration of thrombocytope-
nia (Rodeghiero et al. 2009; Imbach et al. 2006; Ruggeri et al. 2006). In a matched
pair analysis and calculated odds ratios of ICIS Registry I data, the influence of the
initial treatment on the course of ITP was investigated. Assuming that a platelet
count of <50 × 109/l is of more clinical relevance, the matched pair analysis was
repeated with a platelet count of <50 × 109/l as an alternate definition for chronic
ITP and compared with patients with a platelet count of ≥50 × 109/l (Tamminga
et al. 2009). Based on this analysis, a subgroup of children who were treated with
IVIG exhibited a smaller risk of chronic ITP compared with children not receiving
IVIG; it is however not clear which clinical and laboratory factors define such a
subgroup (Tamminga et al. 2009).
19.5 ICIS Registry II (2002–2004)
The observation that a majority of children with newly diagnosed ITP has often no,
mild, or moderate bleeding in spite of a low platelet count stimulated ICIS to design
a cohort study with the aim to test the hypothesis, that major hemorrhage in children
with newly diagnosed ITP is uncommon (Neunert et al. 2008). Major hemorrhage
was defined broadly with Intracranial or other overt internal or mucous membrane
bleeding, which results in anemia or which requires local treatment to stop
hemorrhage. Physical examination included bleeding assessment by using the

bleeding scale of Bolton-Maggs and Moon (1997) and Bolton-Maggs (2003).
Between June 2001 and December 2004, 1106 children were enrolled by 74 inves-
tigators and observed during 2 years at initial presentation, during the first 4 weeks,
at 6, 12, and 24 months. Severe, moderate, and no or mild hemorrhage was found in
3, 20, and 77%, respectively (Neunert et al. 2008). These numbers were almost
identical with those found by Bolton-Maggs in her national survey in the United
Kingdom (Bolton-Maggs and Moon 1997). New bleeding and admission because of
bleeding complications during the 2-year observational time were rare (Neunert
et al. 2013), which was confirmed by others (Rosthøj et al. 2012; Neunert et al.
2014). However, it is important to be reminded that bleeding and its severity in ITP
are difficult to be studied, because of the lack of standardized case definitions, the
difficulties in assessing and reporting bleeding with bleeding scores, and the omis-
sion of reporting bleeding outcomes in many clinical studies, registries, and admin-
istrative databases (Arnold 2015). Prognostic factors for the individual outcome and
for chronic ITP can now be estimated with ICIS Registry I and II data and with other
sources, based on several factors, such as insidious onset of bleeding symptoms,
mild bleeding at initial presentation, a higher platelet count at presentation, age
more than 10 years, and no preceding infectious disorders (Zeller et al. 2005; Glanz
et al. 2008; Revel-Vilk et al. 2013; Heitink-Pollé et al. 2014; Chotsampancharoen
et al. 2017).
19.6 P
ediatric and Adult Registry on Chronic ITP (PARC-ITP)
(Since 2004)
In 2004, ICIS decided to collaborate with adult hematologists, and therefore, a new
global network of investigators involved in basic and clinical science was initiated.
The Pediatric and Adult Registry on Chronic ITP (PARC-ITP) was activated with
the aim to learn more about common and different clinical and laboratory aspects of
ITP in children and adults, to expand our knowledge in the natural history of ITP
and its management, and to gather data which may enable the design of future trials.
The registry has the potential to add modules to the central database. Thus, amend-
ment 1 was added in 2005, which is a DNA bank available for genetic projects in
ITP, and amendment 2 was added in 2008 with the expansion of more clinical ques-
tions including personal and family history of bleeding and thrombotic diseases,
comorbidities of patients, co-medications, and safety surveillance of drug treat-
ment. Data are registered by Internet access (https://www.parc-itp.net/parc-itp/)
with data administration at the ICIS office in Basel, Switzerland. In 2011 an interim
analysis with data of 1784 children and 340 adults with newly diagnosed ITP unex-
pectedly revealed that there have been more common clinical and laboratory find-
ings of children and adults at the time of first presentation, including the bleeding
phenotype and the occurrence of no or mild bleeding, as well as the severity of
thrombocytopenia at first presentation and the likelihood to be treated for bleeding
(Kühne et al. 2011). A second interim analysis is currently undertaken and was
284 T. Kühne
published at the ASH annual meeting in 2016 in preliminary form (Kühne and
Schifferli 2016). The patient population consisted of 3360 children and 420 adults
with newly diagnosed ITP. Follow-up data at 6, 12, and 24 months after the diagno-
sis of ITP were available in 67, 49, and 31% of children and in 77, 64, and 47% of
adults, respectively. Of children and adults with a platelet count of <100 × 109/l at
6 months, 36 and 28% achieved again a remission at 12 months, suggesting that the
potential of recovery, which is observed in children, can also be observed in adult
patients. Patients with persistent and chronic ITP at 6, 12, and 24 months after the
diagnosis of ITP received drug treatment for their ITP in similar frequency in 58,
46, and 47% of children and in 58, 52, and 40% of adults, respectively. The PARC-
ITP Registry will continue to accept new patient data and perform analyses.
19.7 The Splenectomy Registry (Since 1998)
Splenectomy is a successful therapy with the potential to cure ITP (Kaznelson

1916; Kojouri et al. 2004; Aslam et al. 2017). The procedure is not frequently
undertaken in children with chronic ITP, because of the potential of improve-
ment of or even recovery from ITP and of complications of splenectomy including
life-threatening infections caused by encapsulated bacteria and thromboembolic
complications. A further disadvantage of the procedure includes the burden of
life after splenectomy, such as preparing for traveling, reacting when fever
occurs, and taking regular medical controls. Additionally, vaccinations must be
performed prior to surgery and afterwards, as well as antibiotic prophylaxis.
The timing and the management of the splenectomy is not standardized. The
Splenectomy Registry was opened in the early ICIS time of 1998 and contains
data of 357 children from 80 investigators of 27 countries as of February 2017.
The aim of the registry is to learn more about clinical and laboratory character-
istics of children who are planned for splenectomy and to study their manage-
ment and their outcome. An interim analysis of 134 children confirmed that
splenectomy is also successful in pediatric patients with a stable course during
5 years (Kühne et al. 2007). Half of the children were openly splenectomized
and half were splenectomized by laparoscopical technology. More than 86% of
patients responded with a platelet count of >150 × 109/l within 90 days after the
procedure, and another 9% had a partial response. One year later, 80% of the
complete responders maintained their platelet count, and most of them remained
stable during the following 3–4 years. In seven children a sepsis without fatal
outcome was observed. The analysis revealed several aspects of management
including presurgical vaccination and postsplenectomy antibiotic prophylaxis,
time point, and indication of splenectomy, which did not follow current recom-
mendations and guidelines and may be basis for further discussions and attempts
for consensus. There is still a significant lack of clinical data and the indication
and timing of splenectomy remains individually based and controversial
(Schifferli and Kühne 2013).
19.8 Outlook
Clinical research in children and adults with ITP faces several problems: difficulties
and high costs in recruiting patients because of the rarity of the disease, the hetero-
geneous pathomechanisms, which are usually unidentified by routine laboratory,
the diagnostic difficulties with missing laboratory tests to prove ITP, resulting in a
high degree of heterogeneity of study subjects, and the imprecision of treatment
endpoints (e.g., platelet count, bleeding, quality of life, reduction of concomitant
therapies, and patient preferences). Therefore, clinical research and daily practice
differ significantly. These problems may be at least in part resolved by registries and
represent their major advantages: cost-effectiveness, reflection of “real life” of
patients and physicians, ideal tools for the study of rare diseases, international
research with registration of a large number of patients from different origins, and
assessing results, which may serve as a basis for clinical trials. Registries may reveal
information serving as new ideas and focusing on unexpected areas of research. In
most countries registries are feasible with minimal administrative investment.
Internet, software developments, biostatistics, and database technologies underlie
fast-changing processes, and their application and knowledge are the basis for suc-
cessful registries.
In summary the performance of registries represents a success story of ICIS
since 20 years in the attempt to fill the gaps and increase knowledge of ITP in chil-
dren and adults by establishing an international network, bringing physicians and
scientists together to exchange their ideas, and coordinating research with a careful
use of resources.
Acknowledgments The author thanks the numerous ICIS investigators and their patients for pro-
viding their data and for their ongoing support. Many thanks go to Paul Imbach for his invitation
to write this article and for his help. I thank Caroline Martin Asal, Verena Stahel, and Monika
Imbach of the ICIS office in Basel, Switzerland, for their patience and help and all members of the
ICIS Board for their continuous support of our group.
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J Clin Med. 2017;6(2):1–21.
Part IV
Translation from Polyclonal to
Monoclonal Antibody Treatment
Therapeutic Monoclonal Antibodies
as Immunomodulators and Anti-Cancer 20
Agents: Development, Evidence of
Efficacy, Mechanisms of Actions,
Adverse Effects
Tim Niehues
Abbreviations
Abs Antibodies
ACR American College of Rheumatology
ADAs Anti-drug antibodies
ADCC Antibody-dependent cellular cytotoxicity
ADCP Antibody dependent cellular phagocytosis
AEs Adverse events
AML Acute myeloid leukemia)
APC Antigen-presenting cell
BRM Biological response modifiers
CDC Complement Dependent Cytotoxicity
CDCC Complement Dependent Cell mediated Cytotoxicity
CDCP Complement Dependent Cell mediated Phagocytosis
CDRs Complementarity determining regions
CMC Complement dependent cytotoxicity
CSA Ciclosporine A
DAMP Damage-associated molecular pattern
Declaration COI: Tim Niehues is receiving honoraria from Up2date.com as a reviewer and author.
T. Niehues
Zentrum für Kinder- und Jugendmedizin, HELIOS KLINIKUM Krefeld,
Lutherplatz 40, 47805 Krefeld, Germany
Akad. Lehrkrankenhaus, RWTH Aachen, Aachen, Germany

292 T. Niehues
EGFR (synonyms
erbb1, HER1) Epithelial growth factor receptors
ESR Erythrocyte sedimentation rate
EULAR European League against Rheumatism
Fab Variable region of antibody, antigen-binding fragment
FAEs Fatal adverse events
Fc Constant region of antibody, crystalizable fragment
FcR Fc receptors
FcRn Neonatal FC receptor
FDA Food and Drug Administration (USA)
GD2 Glykolipiddisialoganglioside
GMCSF Granulocyte macrophage colony stimulating factor
HAMAs Human anti-mouse antibodies
HAT Medium containing hypoxanthine, aminopterin, thymidine
HER-2 Human epidermal growth factor receptor 2
IBD Inflammatory bowel disease
Ig Immunoglobulin
irAEs Immune related adverse events
ITP Idiopathic thrombocytopenic purpura
IVIG Intravenous immunoglobulins
JAK Janus Kinase
JC virus John Cunningham virus
JiA Juvenile idiopathic arthritis
mAbs Monoclonal antibodies
MAC Membrane attack complex
MS Multiple sclerosis
MTX Methotrexate
NSAIDS Non-steroidal and anti-inflammatory drugs
PAMP Pathogen-associated molecular pattern
PDGFR Platelet-derived growth factor receptor
PEG Polyethylene glycol
PJP Pneumocystis jiroveci pneumonia
PIGF Placental growth factor
PML Progressive multifocal leukoencephalopathy
PRRs Pathogen recognition receptors
RA Rheumatoid arthritis
RANK Receptor activator of nuclear factor kappaB
RANKL Receptor activator of NFκb ligand
SIRs Standard infusion reactions
sJIA Systemic onset JIA
SLE Systemic lupus erythematosus
SYK Spleen tyrosine kinases
TAA Tumor associated antigens
TCR T-cell receptor
TDM Therapeutic drug monitoring
TNF Tumor necrosis factor
Tregs T-regulatory cells
VEGF Vascular endothelial growth factor
20 Therapeutic Monoclonal Antibodies as Immunomodulators and Anti-Cancer Agents 293
20.1 Introduction
Immunotherapy has been a long wish among immunologists. In the late 19th cen-
tury, the first proof of immunotherapy was probably the successful treatment of
diphtheria by Emil von Behring, using serum (Behring and Kitasato 1890). In 1952,
Ogden Bruton showed that patients devoid of immunoglobulins (X-linked agam-
maglobulinemia) can successfully be treated with plasma containing polyclonal
antibodies (Bruton et al. 1952). In 1981, Paul Imbach and colleagues demonstrated
that polyclonal antibody preparations can be used to treat immune-mediated disease
(idiopathic thrombocytopenic purpura (ITP)) (Imbach et al. 1981).
In 1948 Astrid Fagraeus had published the first detailed chemical structure of
antibodies (Fagraeus 1948). In the early 1970s Milstein and colleagues used trans-
formed B-cells (myelomas) and myeloma fusion partners to generate hybrid cells
(so-called hybridomas) that produced large amounts of different antibodies. Georges
Köhler joined Milstein in 1973, immunized mice with sheep red blood-cells and then
fused the lymphocytes from their spleen with myelomas. These hybridomas pro-
duced specific antibodies to sheep red blood-cells with a single type of specific anti-
body termed monoclonal antibodies (mAbs) (Köhler and Milstein 1975). Milstein
and Köhler received the Nobel Prize in 1984 but never patented their findings. In
2014, the top ten therapeutic mAbs sold for annual global sales of more than 72 bil-
lion dollars (https://www.thebalance.com/top-biologic-drugs-2663233). In 2020, it
is estimated that 70 monoclonal antibody products will be on the market with world-
wide sales of nearly 125 billion dollars (Ecker et al. 2015).
20.2 Definitions
Monoclonal antibodies (mAbs) resemble human physiological molecules with high

molecular complexity and belong to the group of biologics. There are different terms
for mAbs used as therapeutic agents: Biological response modifiers (BRM), biolog-
ics, biologicals, therapeutic antibodies, biologic agents, biopharmaceuticals, etc.
Biologics describe any agent that is made by a biological process. According to
FDA definitions biologics are medical products made from living sources (human,
animal, microorganisms), e.g. vaccines, blood products, allergenic extracts, gene
therapies, cellular therapies, etc.
Difference of Biologics to conventional drugs: Conventional drugs consist of
chemical substances with known structures. Biologics are complex mixtures that
are not easily identified or characterized. They tend to be heat-sensitive and suscep-
tible to microbial contamination.
Biosimilars are copies of biologics (see Table 20.1). According to WHO a biosimi-
lar is a biotherapeutic product which is similar in terms of quality, safety, and efficacy
to an already licensed reference biotherapeutic product. It has the same primary amino
acid sequence as the reference product and has undergone rigorous analytic and clini-
cal testing in comparison with its reference product (Anonymous 2009; Chingcuanco
et al. 2016; Kay 2016). Based on sufficient biosimilarity and interchangeability, bio-
similars have been successfully used in patients (e.g., TNFα inhibitors, Etanercept,
Adalimumab, Infliximab, and Rituximab).
294
Table 20.1 Targets for therapeutic morbs on hematopoietic cells other than lymphocytes, licensed by FDA (last updated in 03/2017)
Target system Target molecule Antibody Brand name Type of antibody/protein Route Indication (FDA)
Granulocytes (adhesion molecules)
Alpha-4 integrin Natalizumab Tysabri Humanized IgG4κ i.v. Relapsing forms of
MS, Crohn’s disease
Integrin α4β7 (LPAM-1, Vedolizumab Entyvio Humanized i.v. Ulcerative colitis
lymphocyte Peyer’s patch
adhesion molecule 1)
Platelets
GPIIb/IIIa Abciximab ReoPro Chimeric/human i.v. Cardiac ischemia
monoclonal antibody; Fab
portion
Comments/Meta-analyses (emphasis on Cochrane library, last 10 years):
MS: In relapsing remitting multiple sclerosis Interferons, Natalizumab, Alemtuzumab, Daclizumab, and other drugs were examined. Alemtuzumab,
Natalizumab, and Fingolimod were the best choices of preventing clinical relapse in a 24-month follow-up. For prevention of disability only Natalizumab
showed a beneficial effect (Tramacere, Cochrane Database Syst Rev. 2015 Sep 18;(9):CD011381)
IBD: Controlled randomized trials on moderate to severe Crohn’s disease or ulcerative colitis were examined regarding mucosal healing as an endpoint
(observation periods of 6–12 weeks for induction, 32–54 weeks for maintenance). Anti-TNF was more effective than placebo for maintaining mucosal
healing in Crohn’s disease. In ulcerative colitis Adalimumab was inferior to Infliximab. Both Infliximab and Adalimumab were similar in Crohn’s disease.
(Cholapraneh, Elementary pharmacology and therapeutics 2017;45:1291–1302)
T. Niehues
Table 20.2 Targets for therapeutic mabs licensed by FDA (last updated in 03/2017) on lymphocytes, T-cells and antigen-presenting cells (APC), B cells and
soluble targets (B cell specific cytokines)
Target Target Brand
system molecule Antibody name Type of antibody/protein Route Indication (FDA)
Lymphocyte subpopulation
CD52 Alemtuzumab Campath, Humanized IgG1κ, recombinant i.v. Chronic B-lymphocyte Leukemia
Lemtrada (B-CLL)
T-lymphocytes
IL2RA Basiliximab Simulect Chimeric IgG1κ, recombinant i.v. Prophylaxis of acute organ rejection in
adults
IL2R Daclizumab Zinbryta Humanized s.c. Multiple sclerosis
IL-2R Denileukin ONTAK Recombinant fusion protein expressing i.v. Recurrent CD25 positive, cutaneous T-cell
diftitox amino acid residues of diphtheria toxin lymphoma
fragment A & B, followed by the
sequence for IL-2
CD3/CD19 Blinatumomab Blincyto Mouse, bispecific i.v. Precursor B-cell acute lymphoblastic
leukemia
(continued)
20 Therapeutic Monoclonal Antibodies as Immunomodulators and Anti-Cancer Agents
295
Target Target Brand
296
system molecule Antibody name Type of antibody/protein Route Indication (FDA)

T-lymphocyte/APC (synapse)
B7-1 (CD80), Abatacept Orencia CTLA4-Human IgG1 Fusion protein s.c. Rheumatoid Arthritis, Juvenile
B7-2 (CD86) Rheumatoid Arthritis
CD80, CD86 Belatacept Nulojix CTLA fused to the Fc portion of human i.v. Prophylaxis of organ rejection after
IgG1 kidney transplant
CTLA-4 Ipilimumab Yervoy Chimeric IgG1κ, recombinant i.v. Melanoma that is metastatic or
monoclonal antibody unresectable
PD-1 Nivolumab Opdivo Fully human i.v. Metastatic melanoma, Metastatic
squamous non-small cell lung carcinoma
PD-1 Pembrolizumab Keytruda Humanized i.v. Metastatic melanoma
PD-L1 Atezolizumab Tecentriq Humanized i.v. Urothelial carcinoma
PD-L1 Durvalumab Imfinzi Fully human i.v. Urothelial carcinoma
PD-L1 Avelumab Bavencio Fully human i.v. Metastatic Merkel cell carcinoma
Metastatic melanoma: Various immunotherapy agents alone or in combination with chemotherapy have been used (checkpoint inhibitors: Ipilimumab,
Pembrolizumab, Nivolumab). No systematic Cochrane reviews available
Metastatic non-small cell lung carcinoma: Nivolumab, pembrolizumab have been used as some of the tumors overexpress PDL1.ipilimumab as inhibitor
of CTLA4. No systematic Cochrane reviews available
Urothelial carcinoma: Atezolizumab has been used in metastatic disease. No systematic Cochrane reviews available
B cells and B cell specific cytokines
BLyS Belimumab Benlysta Human monoclonal antibody i.v. Systematic lupus erythematosus (SLE)
(BAFF) IgG1-lambda
CD20 Ocrelizumab Ocrevus Humanized i.v. Systemic lupus erythematosus (SLE),
Multiple sclerosis (MS)
CD20 Rituximab Rituxan Chimeric IgG1κ, recombinant i.v. Rheumatoid arthritis; B-cell non-
Hodgkin’s lymphoma
SLE: Belimumab showed a significant benefit compared to standard-of-care treatment in patients with primary cutaneous and musculoskeletal
T. Niehues
manifestations of SLE (Navarra SV, Lancet 2011;377:721; Furie, Arthritis and Rheumatism 2011;63:3918). Atacicept, a fusion protein blocking BLYS and
APRIL showed no difference to placebo at a standard dose (Isenberg, Annals of rheumatic diseases 2015;74:2006)
Table 20.3 Therapeutic mabs and fusion proteins against tumor necrosis factor (TNF), licensed by FDA (last update 03/2017)
Target
molecule Antibody Brand name Type of antibody/protein Route Indication (FDA)
TNF Adalimumab Humira Human IgG1κ, recombinant s.c. Rheumatoid arthritis; juvenile idiopathic arthritis;
psoriatic arthritis; ankylosing spondylitis; Crohn’s
disease; plaque psoriasis
TNF Certolizumab Cimzia Humanized, recombinant Fab’ s.c. Crohn’s disease
pegol fragment conjugated to
polyethylene glycol
TNFα and Etanercept Enbrel TNFR-IgG1 Fc fusion protein s.c. Rheumatoid arthritis; juvenile idiopathic arthritis;
TNFβ psoriatic arthritis; ankylosing spondylitis; Crohn’s
disease; plaque psoriasis
TNF alpha Infliximab Remicade Chimeric IgG1k, recombinant i.v. Rheumatoid arthritis (in combination with methotrexate);
monoclonal antibody Ankylosing spondylitis; Psoriatic arthritis; Plaque
psoriasis; Crohn’s disease; Ulcerative colitis; Pediatric
ulcerative colitis
TNF Infliximab- Inflectra Chimeric, biosimilar i.v. Crohn’s disease, Ulcerative colitis, Ankylosing
dyyb spondylitis, Psoriatic arthritis, Plaque psoriasis
TNF Golimumab Simponi Aria Fully human i.v. Rheumatoid arthritis
(continued)
297
298
Target
TNF Golimumab Simponi Human IgG1κ, recombinant s.c. Rheumatoid arthritis; Psoriatic arthritis; Ankylosing
spondylitis
TNF Adalimumab- Amjevita Fully human, biosimilar s.c. Rheumatoid arthritis
atto
TNF Infliximab- Renflexis Chimeric, biosimilar i.v. Crohn’s disease, Ulcerative colitis, Rheumatoid arthritis,
abda Ankylosing spondylitis, Psoriatic arthritis, Plaque
psoriasis
Rheumatoid arthritis RA: Abatacept, Adalimumab, Etanercept, Infliximab, and Rituximab were associated with significant improvement in the ACR 50
rate. Anakinra was less effective than all the other biologics. (Singh CMAJ, Canadian Medical Association Journal 2009;181:787). Patients were more
likely to withdraw from the trials if they were on biologics. There is no evidence that any one of the TNF inhibitors has greater efficacy than the others
(Medical Letters drugs therapy 2010:52(1338):38)
Ankylosing spondylitis: TNF inhibitors increase the chance of achieving partial remission and slight improvement of spinal inflammation as measured by
MRI. (Maxwell, Cochrane Database Syst Rev. 2015 Apr 18;(4):CD005468). In the biologics group more patients dropped out of the studies because of
side-effects. Compared to placebo, improvement with TNF blockers was rated between 25 and 40%. Partial remission was reached in 10–44%. TNFα
blockers were shown to improve disease activity and functional capacity in a clinically meaningful manner (Callhoff et al. Annals of Rheumatic Diseases
2015;74:1241)
JIA Polyarthritis: The majority of trials show a flawed trial design (so-called withdrawal design). The only properly designed double blind randomized
placebo controlled trial did not show a significant effect regarding efficacy of Infliximab (Ruperto, Arthritis and Rheumatism 2007;56:3098)
Pediatric psoriasis: There is a single randomized controlled trial (industry sponsored and observation of 12 weeks) Sanclemente, Cochrane Skin Group
2015 DOI: 10.1002/14651858.CD010017.pub2)
T. Niehues
Table 20.4 Therapeutic mabs and fusion proteins against Interleukin 1 (IL 1) and Interleukin 6 (IL 6), licensed by FDA (last update 03/2017)
Target
system Target molecule Antibody Brand name Type of antibody/protein Route Indication (FDA)
Interleukin 1
Interleukin-1 Anakinra Kineret Recombinant, s.c. Moderately to severely active rheumatoid
type I receptor nonglycosylated form of arthritis, in patients 18 years of age or older
(IL-1RI) the human interleukin-1 who have failed 1 or more disease modifying
receptor antagonist antirheumatic drugs
(IL-1Ra)
IL1B Canakinumab Ilaris Human anti-human-IL-1β s.c. Treatment of CAPS, including Familial Cold
IgG1 monoclonal antibody Autoinflammatory Syndrome (FCAS), and
Muckle-Wells Syndrome (MWS), in adults
and children 4 years of age and older
IL-1 beta Rilonacept Arcalyst Dimeric fusion protein; s.c. Cryopyrin-associated periodic fever
(IL-1β) ligand-binding domains of syndromes (CAPS), including Muckle-Wells
IL-1 receptor (IL-1R) & syndrome (MWS) and familial cold
IL-1 receptor accessory autoinflammatory syndrome (FCAS) in
protein (IL-1RAcP) linked children 12 years and older
to human IgG1
Interleukin 6
IL6 Siltuximab Sylvant Chimeric i.v. Multicentric Castleman’s disease
IL6R Tocilizumab Actemra Humanized i.v., s.c. Rheumatoid arthritis, Polyarticular juvenile
idiopathic arthritis, Systemic juvenile
idiopathic arthritis
RA: The Il1-blocking Anakinra was less effective than all the other biologics. (Singh, Canadian Medical Association Journal 2009;181:787)
Crohn’s disease: A Tocilizumab trial has been stopped as a number of bowel perforations were observed that might have been obscured by the fact that
CRP levels cannot be judged in antiIL6R treatment because CRP levels depend on IL6
Castleman’s disease: Is due to an excessive release of pro-inflammatory cytokines and excess proliferation of B-and T-cells. Targeting IL6 may be a
promising approach to treat HHV8 negative multicentric Castleman’s disease

299
Table 20.5 Therapeutic mabs against IgE, Interleukin 4 (IL4) and Interleukin 5 (IL5), licensed by FDA (last update 03/2017)
300
Type of antibody/
Target system Target molecule Antibody Brand name protein Route Indication (FDA)
IgE
IgE Omalizumab Xolair Humanized IgG1κ, i.v. Moderate to severe IgE-mediated, persistent
recombinant asthma (US) or severe asthma (EU), in adults
and children over 12 years old, inadequately
controlled by inhaled corticosteroid treatment
Interleukin 4
IL4RA Dupilumab Dupixent Fully human s.c. Atopic dermatitis
Interleukin 5
IL5 Mepolizumab Nucala Humanized IgG1κ s.c. Severe eosinophilic asthma in patients aged
monoclonal antibody 12 years or older
IL5 Reslizumab Cinqair Humanized i.v Severe asthma
Asthma: Omalizumab was effective in reducing asthma exacerbations and hospitalizations as an adjunctive therapy to inhaled steroids and during steroid
tapering phases of clinical trials. It remains to be tested prospectively whether the addition of omalizumab has a prednisolone-sparing effect and whether
there is a threshold level of baseline serum IgE for optimum efficacy of omalizumab. Given the high cost of the drug, identification of biomarkers
predictive of response is of major importance for future research. (Normansell, The Cochrane Library: 13 January 2014; DOI: 10.1002/14651858.
CD003559.pub4)
Severe eosinophilic asthma: There is a single study on Mepolizumab in participants with severe eosinophilic asthma: improvement in health-related quality
of life scores and reduction of asthma exacerbations. There are no studies reporting results from children. (Powell, Cochrane Database of Systematic
Reviews 2015, Issue 7. Art. No.: CD010834. DOI: 10.1002/14651858.CD010834.pub2))
T. Niehues
Table 20.6 Therapeutic mabs against Interleukin 17 (IL 17), Interleukin 12 (IL 12), and Interleukin 23 (IL 23), licensed by FDA (last update 03/2017)
Type of antibody/
Interleukin 17
IL17A Ixekizumab Taltz Humanized s.c. Plaque psoriasis
IL17A Secukinumab Cosentyx Human s.c. Moderate to severe plaque
immunoglobulin G1k psoriasis; ankylosing
(IgG1k) subclass spondylitis; psoriatic
monoclonal antibody arthritis
IL17RA Brodalumab Siliq Chimeric s.c Plaque psoriasis
Interleukin 12, Interleukin 23
IL12 Ustekinumab Stelara Human IgG1k s.c Adult patients (18 years or
monoclonal antibody older) with moderate to
severe plaque psoriasis who
are candidates for
phototherapy or systemic
therapy
Severe Crohn’s disease: Ustekinumab was not significantly better than placebo in moderate to severe Crohn’s disease (Rogler, Digestive diseases
2017;35:5–12)
Psoriasis: Biologics are highly effective for the treatment of moderate to severe psoriasis, and anti-IL-17 drugs have the same, or even greater, efficacy
than anti-tumor necrosis factor (TNF) and anti-IL-12/23 drugs. (de Carvalho, Drugs R D. 2017 Mar; 17(1): 29–51)
301
Table 20.7 Targets on vessels and bone as well as soluble targets (complement) for therapeutic mabs and fusion proteins, licensed by FDA
302
(last update 03/2017)

Target Target
system molecule Antibody Brand name Type of antibody/protein Route Indication (FDA)
Complement
Complement Eculizumab Soliris Humanized i.v. Paroxysmal nocturnal hemoglobinuria
component 5
Vessels: Vascular and placental growth factors and receptors
VEGF, PIGF Aflibercept Eylea Fusion protein: IgG Fc, and Intravitreal Wet age-related macular edema; Macular
ligand binding domains of injection edema following central retinal vein
VEGFR-1, VEGFR-2 occlusion
VEGF Bevacizumab Avastin Humanized monoclonal i.v. Metastatic colorectal cancer;
antibody unresectable, locally advanced, recurrent
or metastatic non-small cell lung cancer
VEGFR1 Ranibizumab Lucentis Humanized IgG1κ Fab Intravitreal Neovascular (wet) age-related macular
fragment injection degeneration
VEGFR2 Ramucirumab Cyramza Fully human i.v. Gastric cancer
VEGF, PIGR Ziv-aflibercept Zaltrap Fusion protein: IgG Fc, and i.v. Metastatic colorectal cancer
ligand binding domains of
VEGFR-1 and VEGFR-2
Bone: osteoclast activation
RANKL Denosumab Prolia, Xgeva Human IgG2 monoclonal Subcutaneous Xgeva: Prevention of skeletal-related
antibody events in patients with bone metastases
from solid tumors; Prolia: treatment of
postmenopausal osteoporosis; treatment
of men receiving androgen deprivation
Metastatic colorectal cancer: Bevacizumab has varying success
Metastatic non-small cell lung carcinoma: Addition of Bevacizumab to Carboplatin and Paclitaxel may increase overall survival and progression-free
survival in some patients (Sandler, NEJM 2006;355:2542)
T. Niehues
Gastric cancer: Ramucirumab has been used in cases of advanced gastric cancer. It is unclear how effective it is
Table 20.8 Tumor associated antigenes (TAA) and growth factor receptors as targets for therapeutic mabs, licensed by FDA (last update 03/2017)
Target
HER2 Ado- Kadcyla Humanized IgG1κ mAb i.v. HER-2 positive metastatic breast cancer
trastuzumab
emtansine
EGFR Cetuximab Erbitux Chimeric monoclonal antibody i.v. Metastatic colorectal cancer, head and neck cancer
GD2 Dinutuximab Unituxin Chimeric i.v. Pediatric high-risk neuroblastoma
EGFR Panitumumab Vectibix Recombinant human IgG2κ i.v. Treatment of EGFR-expressing, metastatic
monoclonal antibody colorectal carcinoma with disease progression on or
following fluoropyrimidine-, oxaliplatin-, and
irinotecan- containing chemotherapy regimens
HER2 Pertuzumab Perjeta Humanized IgG1κ monoclonal i.v. HER2 overexpressing breast cancer, in combination
antibody, glycosylated with trastuzumab and docetaxel
PDGFRA Olaratumab Lartruvo Fully human i.v. Soft tissue sarcoma
HER2 Trastuzumab Herceptin Humanized receptor antagonist i.v. HER2 overexpressing breast cancer
EGFR Necitumumab Portrazza Fully human i.v. Metastatic squamous non-small cell lung carcinoma
Breast cancer: Trastuzumab has improved 5-year disease-free survival in tumors overexpressing the receptor HER2 significantly
Neuroblastoma: Dinutuximab and addition of cytokines GMCSF, IL2 and Isotretinoin improves outcome (Yu, NEJM 2010)
Metastatic colorectal cancer: Cetuximab and Panitumumab have varying success
Soft-tissue sarcoma including gastrointestinal tumors: Studies have focused on inoperable metastatic and/or recurrent disease using Olaratumab either
with Doxorubicin or without
303
Table 20.9 Targets on hematopoietic malignant cells, licensed by FDA (last update 03/2017)
304
Target Target
system molecule Antibody Brand name Type of antibody/protein Route Indication (FDA)
CD38 Daratumumab Darzalex Fully human i.v. Multiple myeloma
SLAMF7 Elotuzumab Empliciti Humanized i.v. Multiple myeloma
CD19 Blinatumomab Blincyto Mouse, bispecific i.v. Precursor B-cell acute lymphoblastic
leukemia
CD30 Brentuximab Adcetris Chimeric IgG1k monoclonal i.v. Hodgkin’s lymphoma, Systemic
vedotin antibody conjugated to a microtubule anaplastic large cell lymphoma
disrupting agent MMAE by a
protease-cleavable linker
CD20 Ibritumomab Zevalin Murine IgG1κ monoclonal antibody, i.v. Non-Hodgkins Lymphoma (CD20
tiuxetan conjugated to a chelator (tiuxetan) positive, low-grade or follicular) which
for labeling with Indium-111 or is relapsed or refractory to rituximab, in
Yttrium-90 combination with rituximab
CD20 Tositumomab Bexxar Murine, monoclonal; covalently Non-Hodgkins Lymphoma (CD20
bound to Iodine-131 positive, follicular) which is refractory to
rituximab and relapsed following
chemotherapy
CD20 Obinutuzumab Gazyva Humanized IgG1 monoclonal i.v. Chronic Lymphocytic Leukemia in
antibody combination with chemotherapy in
treatment-naïve patients
CD20 Ofatumumab Arzerra Human IgG1κ monoclonal antibody, i.v. Chronic lymphocytic leukemia resistant
recombinant to fludarabine and alemtuzumab
Lymphoma B-cell chronic lymphocytic leukemia, Non-Hodgkins Lymphoma, Hodgkins Lymphoma and Anaplastic large-cell lymphoma: Rituximab has
been used for B-cell Non-Hodgkins Lymphoma and is included in current chemotherapy standard chemotherapy protocols as it has been shown to be
effective (Chop-R). Brentuximab has been used in Hodgkin lymphoma but is not part of the standard chemotherapy protocols
Chronic Lymphocytic Leukemia (CLL): Obinutuzumab and Ofatumumab, Alemtuzumab have been used but are not part of the standard protocols.
Rituximab plus chemotherapy is more effective than chemotherapy alone. (Bauer, Cochrane Library 2012, DOI: 10.1002/14651858.CD008079.pub2)
T. Niehues
Multiple Myeloma High-dose chemotherapy with autologous hematopoietic stem cell transplantation has become the treatment of choice in patients under
65 years of age. In relapses Elotuzumab and Daratumumab have been tried
Table 20.10 Targets for therapeutic mabs on microorganisms, liver cells, and drugs licensed by FDA (lcs update 3/2017)
Type of antibody/
Microorganisms
F protein of RSV Palivizumab Synagis Humanized IgG1κ Intramuscular Prevention of serious lower respiratory
tract disease caused by respiratory
syncytial virus (RSV) in pediatric
patients at high risk of RSV disease
Protective antigen of Raxibacumab Raxibacumab Human IgG1- i.v. Inhalational anthrax
Bacillus anthracis lambda mAB
Protective antigen of Obiltoxaximab Anthem Chimeric i.v. Inhalational anthrax
the Anthrax toxin
Clostridium difficile Bezlotoxumab Zinplava Fully human i.v. Prevent recurrence of Clostridium
toxin B difficile infection
Liver cells/LDL-receptors
PCSK9 Evolocumab Repatha Fully human s.c. Heterozygous familial
hypercholesterolemia; Refractory
hypercholesterolemia
PCSK9 Alirocumab Praluent Fully human s.c. Heterozygous familial
hypercholesterolemia; Refractory
hypercholesterolemia
Drugs
Dabigatran Idarucizumab Praxbind Humanized Fab i.v. Emergency reversal of anticoagulant
dabigatran
305
306 T. Niehues
Kinase inhibitors are relative simple chemical compounds, having Janus (JAK)
and Spleen tyrosine kinases (SYK) as targets. Kinase inhibitors may result in simi-
lar effects as they may target the same pathway as a monoclonal antibody. Unlike
biologics they are not proteins and hence not biologics. They may have similar
effects on immune-mediated diseases as many mAbs, as some of them inhibit the
downstream signaling of proinflammatory cytokines (Patterson et al. 2014; Wu
et al. 2015). In this article I focus on mAbs, biosimilars, and engineered proteins
(e.g., fusion proteins, bispecific antibodies) that have entered clinical practice.
20.3 Terminology
The USAN (United States Adopted Names) Council has proposed guidelines for the
naming of mAbs (https://www.ama-assn.org/about/monoclonal-antibodies).
Starting from the end of the name all monoclonal antibodies end with the suffix –
mab (Fig. 20.1) (Ballow 2005). Any monoclonal antibody name is composed of
combining key elements
1. Prefix
2. Infix, representing the target or disease and/or indicating the source
3. Stem used as suffix
Ante
Prefix Penultimate Penultimate
to create Syllable to syllable to
unique indicate target/ indicate Ultimate
name disease class source syllable
ada -lim -u -mab
-tu/-t = tumours -u = fully human -mab = monoclonal

antibody
-lim/-li = immuno -o = mouse
modulation -cept = receptor
-hu = humanized fusion protein
-ba/-b = bacterial
-xi = chimeric
-ci/-c = cardiovascular
-fu / -f = fungal
-ki / -k = interleukins
-ne / -n = neurons
-bo / -b = bone
-vi / -v = viruses
Fig. 20.1 Nomenclature INN (International non-priority names) for biologicals and biotechnol-
ogy substances. Adapted from www.ama-assu.org/about monoclonal-antibodies
Prefixes are used to create a unique name, a distinct compatible syllable as the
starting prefix. The choice of inflix is determined by the available information
regarding clinical indications and antibody action. The target disease inflix has been
truncated with a single letter when the source inflix begins with a vowel. Examples
for target/disease class inflixes are shown in Fig. 20.1.
Recently, it has become quite difficult to capture the mAbs vowel in a single syl-
lable, so terminology is changing (Parren et al. 2017).
20.4 Antibody Structure and Engineering
The basis for development of therapeutic antibodies is the precise knowledge of the
normal IgG antibody structure and activity, which is schematically outlined in
Fig. 20.2a. IgG can be engineered to a higher specificity, longer half-life, and better
bio-availability (summarized in Fig. 20.2b) The function of antibodies is dependent
on their binding affinity and target specificity. Instability or aggregation hotspots in
the antibodies CDRs can be removed, the hinge can be stabilized and glycosylation
inhibited (e.g., aglycosylated IgG4). In the variable region (Fab), affinity can be
altered and may allow targeting antigens even more selectively.
The Fc region of any antibody interacts with immune cells carrying Fc receptors
(FcR) (e.g., macrophages). By engineering glycosylation status antibody dependent
cellular cytotoxicity can be altered. The half-life of the antibody can be engineered
by altering binding of the Fc part of the mAb to the neonatal FC receptor (FcRn)
(e.g., on endothelial cells). MAbs can be associated with highly cytotoxic drugs,
cytokines, toxins, etc.
Bispecific antibodies with a simultaneous blockade of two or more targets may
be even more promising than inhibition of a single target (e.g., the bispecific anti-
body Blinatumomab).
20.5 Production Technology
MAbs are typically made by fusing myeloma cells with mouse spleen cells that
have been previously immunized with the desired antigen and future target.
Myeloma cells have lost the ability to grow in standard media. This can be exploited
by supplementing standard culture media with so-called HAT medium containing
hypoxanthine, aminopterin, and thymidine and thus selectively grow hybridomas.
Single cell clones are grown on microtiter wells and the antibodies secreted by dif-
ferent clones are assayed for their ability to bind antigen. The most productive and
stable clones are selected for future use. Injected into mice, these hybridomas can
produce antibody-rich fluid (ascites), containing large amounts of monoclonal anti-
body. In 1986, the first murine monoclonal antibody Muromonab (CD3 specific)
was used clinically. Initial therapeutic antibodies were composed of all mouse pro-
tein which were highly immunogenic leading to human anti-mouse antibodies
(HAMAs) and toxicities.
Moreover, mAbs are heterogeneous as they contain aggregates, degradation
products, glycosylation variants, oxidized side chains, as well as amino and c arboxyl
308 T. Niehues
a VH CDR
VH
VL VL
Fab region
(antigen binding) C H1 CH1
CL CL
FcR mediated
Disulphide bonds effector functions
Glycosylation Hinge
Sialic Acid CDCP
Galactose
N-Acetyl- C1q CDCC
CH2 CH 2
Glycosamine
Mannose Makrophage ADCP
Fc
Asn 297 region Fc R
Fucose (CD16) NK cells ADCC
CH3 CH 3 (CD32)
FcRn (CD64)
Maintenance of serum IgG

e.g. Movement of IgG
endothelial between compartments
cell (e.g. placental transfer)
Transcytosis
b Increase stability and homogeneity Improve target specificity

• Selection of amino-terminal glutamine • Modulation of antigen
residues to force pyroglutamylation specificity and affinity
• Decrease number of charge variants • Restore Ag-binding
Increase stability
• Mutation of instability hotspots
in CDRs
Fab regions
Decrease aggregation Shorten/increase serum half live

• Introduction of cysteine residues • Fab monovalent format
mutation of other amino acids • Fab-PEG
Decrease immunogenicity
• Humanization and de-humanization
Limit number of structural
isomers Fc region
• IgG2/IgG4 hinge engineering, (effector Improve effector function
avoid half IgGs, disulphide function)
bridge isomers Link isotope with
instable link
Link drug with
instable linker
Link toxins, cytokines,
Glycoengineering (select enzymes
advantageous glycoforms)
• Aglycosylation ADCC ,
Shorten/increase serum half live
ADPC , CDC Increase stability and homogeneity
• Bisecting N-acetyl • Deletion of carboxy terminal lysine • Use different isotype e.g. IgG4
glucosamine ADCC residues • Mutations in Fc region
• Non-fucosylation ADCC • Decrease number of charge variants • Delete entire Fc region
Improvement/modulation of pharmaceutical progerties is depicted in green; Improvement of antibody function in red and Modulation
of pharmacokinetics/pharmacodynamics in blue
amino acid additions. All of these can have consequences for product potency,
bio-availability, and immunogenicity. Moreover, mAbs do not experience the

post-translational modification that physiological abs get from mammalian cells
(e.g., glycosylation).
In 1984, antibody chimerization had been described by Morrison et al. (1984).
Chimeric antibodies had been created by combining murine monoclonal antibody
variable regions with the constant region of a human antibody. In 1986, antibody
humanization was described by Jones et al. (1986). Humanized mAbs were devel-
oped which incorporated only the hypervariable or complementarity determining
regions (CDRs) of the murine monoclonal antibody to the remaining portions of the
human IgG molecule. Such a humanized antibody then had less than 10% of their
murine constituent.
In the early 1990s Phage display technology, single B-cell cultures, and usage of
transgenic mice allowed for fully humanized and then fully human antibodies (Beck
et al. 2010).
20.6 Pharmacokinetics
From polyclonal IgG preparations it is known that IgG has a half-life of 3–4 weeks.
The half-life will be influenced by lysosomal degradation that can be prevented by
binding to a scavenger receptor, the neonatal FC receptor (FcRn), e.g. expressed in
endothelial cells. The bio-distribution of mAbs was studied by using radio-labelled
mAbs and measuring their distribution by positron emission tomography PET:
mAbs are cleared gradually from the circulation by liver, spleen, and kidneys and
specifically accumulate at the target site. MAbs even are located to the human brain,
which originally was thought to be a sanctuary (Oosting et al. 2015).
Fig. 20.2 (a) Normal IgG 1 structure and its interaction at the Fc region with complement and effector
cells via different Fc receptors (e.g. Fc γ receptor, Fc receptor n) (b) Engineering of IgG1. Adapted from
Beck et al. (2010), Chan and Carter (2010), and Weiner (2015). Illustration: Oliver Hippmann,
Schwarzenbruck. ADCC = antibody dependent cellular cytoxicity; ADCP = antibody dependent cellular
phagocytosis; Asn = Asparagine at position 297; CD16 = FcγR on NK cells, mast cells, basophils, DC;
CD32, CD64 = FcγR on Makrophage, neutrophils, eosinophils, DC; CDC = complement dependent
cytotoxicity; ;CDCC = complement dependent cell mediated cytotoxicity; CDCP = complement depen-
dent cell mediated phagocytosis; CDR = complementarity determining region; CL1–3 = constant region
light chain 1–3; CH1–3 = constant region heavy chain 1–3; Fab = fragment antigen binding; Fc = frag-
ment crystallizable; FcR = Fc receptor; FcRn = neonatal Fc receptor; MAC = membrane attack com-
plex; PEG = polyethylen glycol; VL = variable region light chain; VH = variable region heavy chain
310 T. Niehues
20.6.1 Factors Influencing Pharmacokinetics
Pharmacokinetics is influenced by significant inter-patient variability (Oude

Munnink et al. 2016). It is influenced by demographic variables (body weight, body
surface area, gender). Most mAbs are dosed based on body weight or body surface
area to equalize monoclonal antibody exposure between patients. As circulating
plasma volume is not linearly correlated with body weight, it has been suggested to
use fixed dosing instead of weight or surface adapted dosing to reduce variability in
exposure. For the monoclonal antibody Panitumumab, Rituximab, Bevacizumab,
and Infliximab clearance in females was 23–39% lower and distribution volume
14–22% smaller compared to males. In other studies, this effect was not shown, so
dosing of mAbs is still gender-independent and bodyweight/surface dependent.
There is no specific clearance and subsequent degradation. Pharmacokinetics is
dependent on disease activity. After having entered the interstitial space and spe-
cific binding to the target antigen has taken place, the antigen immune complex is
prone to FcR mediated phagocytosis by immune cells and clearance. Target-
mediated degradation is dependent on target antigen availability which is likely to
be higher in active or non-controlled disease, e.g. in patients with active inflamma-
tory bowel disease or RA, e.g. high levels of TNFα are associated with an increased
clearance of Infliximab. Interestingly, in the Atlas Study Infliximab and Adalimumab
concentration in tissue biopsies correlated with serum concentrations of the TNFα
antibodies. The better the correlation was between serum and tissue antibody con-
centrations, the more likely patients were in endoscopic remission of their inflam-
matory bowel disease. High tissue levels of TNFα appear to act as a sink for
monoclonal anti TNFα antibodies (Yarur et al. 2016). Similarly, a high tumor load
in lymphoma patients is associated with low Rituximab serum concentrations
(Golay et al. 2013).
Immunogenicity may limit bioavailability and function of therapeutic mAbs.
The less human the therapeutic antibody is, the more likely is the generation of anti-
drug antibodies (ADAs). In a study with Infliximab in inflammatory bowel disease
ADAs were found in a third of the patients and were associated with high Infliximab
clearance (Dotan et al. 2014).
Drugs coadministered with mAbs may delay the clearance of mAbs, e.g. intrave-
nous immunoglobulins (IVIG), which may saturate Fc receptors on endothelial cells/
macrophages/monocytes/tissue macrophages. Co-administration of Methotrexate
with anti-TNF antibodies like Infliximab and Adalimumab seems to be advantageous
because Methotrexate may significantly lower ADAs induction. Lastly, the route of
administration is influencing drug levels as subcutaneous administration only appears
to have 50–80% bioavailability.
20.6.2 Therapeutic Drug Monitoring TDM
As a consequence, it appears to be logical to do therapeutic drug monitoring in

patients treated with mAbs, which is routinely applied in conventional drugs like
antibiotics, anti-epileptics, immunosuppressive drugs, etc. The rationale of TDM is

to correlate serum concentration and response in order to document interpatient vari-
ation in pharmacokinetics, to define the therapeutic window and to allow for flexibil-
ity in dosing. Subtherapeutic monoclonal antibody concentrations may result in
disease progression which in turn means increased target antigen expression calling
for a higher dosage of therapeutic monoclonal antibody in patients with active dis-
ease. This vicious cycle may be associated with lowered response and prospective
randomized clinical trials have been initiated to explore whether systematic TDM
may have a place in treatment algorithms of inflammatory and malignant diseases
treated with mAbs.
20.6.2.1 TDM in Immune Mediated Diseases

Antibody trough levels have been estimated for Adalimumab, Infliximab in rheuma-
tologic and inflammatory bowel diseases in several studies. For Adalimumab trough
level cut-offs have been 5–8 mg/l in Rheumatoid Arthritis (RA), 0.3–8.1 mg/l in
Inflammatory Bowel Disease (IBD). For Infliximab, the cut-off-level has been 1–2.5
mg/l in RA and 0.5–6.2 mg/l in IBD.
20.6.2.2 TDM In Oncology Diseases

Only a few studies have been done to determine trough levels of mAbs and clinical
response. For Rituximab the cut-off for trough levels was as variable as 0.3-8.1 mg/l
in B-cell lymphoma. In malignant disease the amount of monoclonal antibody nec-
essary to control the tumor might be quite variable. In most of the studies convinc-
ing exposure—response relationships provide encouraging evidence for the further
exploration of therapeutic drug monitoring TDM.
20.7 Targets
20.7.1 Targets in Immune Mediated Diseases
Targets of mAbs used in the treatment of immune mediated diseases are shown in
Fig. 20.3. The inflammation starts with recognition of any antigen (be it antigen,
tumor antigen, and allergen) by cells of the innate immune system and amplification
of antigen recognition signals by specialized receptors (PRRs: pathogen recognition
receptors, e.g. TOLL-like receptors). PRRs are activated by ligands that have a
damage-associated molecular pattern DAMP or have a pathogen-associated molecu-
lar pattern PAMP (e.g., DNA, RNA from damaged cells or lipopolysaccharides LPS
on microorganisms). PRRs are expressed on tissue macrophages and other efficient
professional antigen-presenting cells. After decades of research, the events of the ini-
tial pathological immune activation are still unclear in many diseases. If setting and
exact nature of autoantigens would be known it may be possible to intervene earlier in
the disease process by antigen-specific regulation (e.g., by vaccinations). As of now,
targets of biologics are mainly downstream of the induction at the stage of perpetua-
tion of chronic inflammation or tissue/organ destruction as depicted in Fig. 20.3.
DAMP/PAMP I. Induction of
312
IMMUNOLOGIC SYNAPSE
APC Activation T-cell antigen
Auto-Ag/Tumor-Ag/ HLA induced
Allergen Peptide TZR 1
Muromonab** inflammation
Daclizumab Naive T-cell CD28 2
HLA CD80/86
Basiliximab CD3 CTLA-4
IL2R
CD3/TCR PDL1 PD1
1
Alefacept ** CD2 TGN 1412**
Atezolizumab
2 APC Avelumab Nivolumab
CD52
(e.g. Makro- Durvalumab Pembrolizumab
Alemtuzumab Peptide phage)
Costimulation Abatacept
Belatacept Ipilimumab
Belimumab II. Self-

Atacicept * Atacicept * Dupilumab IL2
IL2 IL12 IL1 Perpetuation
BAFF APRIL IL4R TH2 cells IL4 IFNγ IL2 IL6 of chronic
IL25 IL18 TGF IL23 inflammation
BAFF-R IL13R IL33 IL27 TGFβ
PROINFLAMMATORY CYTOKINES
Taci Retinoic acid
Blinatumomab IFNα/β TLR2 IL1β Adalimumab, Rilonacept, Canakinumab
CD19 IL6 Siltuximab, IL6R Tocilizumab
Epratuzumab* B-cells IL4 Ustekinumab Tregalizumab* Ustekinumab
IL21 Aldes- TNFα
CD22 IL12R CD4 leukin (IL2) IL23R Adalimumab, Adalimumab-atto***,
CD20 Mepolizumab TH1 cells Treg IL2R TH17 cells Certolizumab pegol,
IL5
Rituximab Reslizumab Infliximab, Infliximab-dyyb***, Infliximab-abda***,
Ibritumomab Golimumab, Etanercept
Tiuxetan GM-CSF
Ocrelizumab Proinflam- Antiinflam- Proinflam-
Tositumomab matory matory matory
Obinutuzumab Ixekizumab
IL17A, IL17F Secukinumab
Ofatumumab ALLERGY Eosinophil
Plasma cells III. Tissue

Asthma, Inflamed tissue IL17R destruction
Denosumab
Ekzema, (e.g. joint) (e.g. arthritis, CNS
Rhinitis RANK-L Broda- and skin inflam-
RANK limumab Neutrophils mation, vaskulitis)
Osteoclast
IgE
Auto-Abs α4 α4 β7
Omalizumab Bone Natalizumab Vedolizumab
Efalizu- CD11a
C5a Complement VCAM mab** ICAM MADCAM * not approved yet
activation ** withdrawn from market
Eculizumab Synoviocytes Synovitis Endothelium of vessels in inflamed tissues *** biosimilar
T. Niehues
20.7.2 Targets in Oncology
Targets in cancer immunotherapy are shown in Fig. 20.4.
20.7.2.1 Targeting of Tumor Associated Antigens (TAA)

Tumor associated antigens (TAA) represent soluble factors and receptors that
are overexpressed in tumors and are optimal blocking targets to avoid can-
cer cell proliferation, e.g. Epithelial growth factor receptors (EGFR (synonyms
erbb1, HER1)), Vascular endothelial growth factor (VEGF), Placental growth
factors (PLGF1 and PLGF2), Platelet-derived growth factor receptor (PDGFR),
Glykolipiddisialoganglioside (GD2), Receptor activator of NFκb ligand (RANKL)
as well as antigens on cells of the hematopoietic lineage etc.
20.7.2.2 Enhancement of Tumor Immunity

The anticancer immune response or inflammation is complex and compartmental-
ized in blood, lymph nodes, and tumors. Its success depends on priming and activa-
tion of anticancer T-cells, the rate of migration of anticancer T-cells from the lymph
node to the blood and the rate of T-cell infiltration into the tumor as well as the rate
of T-cell efflux from tumors to blood (Chen and Mellman 2017). The so-called
checkpoint inhibitors target T-cell activation within the immunological synapse
(PD1, PD ligand 1, CTLA4). These checkpoint inhibitors prevent effector T-cells
(that fight cancer successfully) from getting exhausted and hyperexhausted (Figs. 20.3
and 20.4). An effective amplifier of the immune system is the use of bispecific anti-
bodies like Blinatumomab, an anti-CD3/anti-CD19 bispecific antibody which simul-
taneously binds tumor antigen (CD19) and recruits T-cells (CD3). Similar approaches
are thought of in neuroblastoma and osteosarcoma (Majzner et al. 2017).
20.8 Mechanism of Action
There are five non-overlapping mechanisms described. Clearly some therapeutic

mAbs use multiple mechanisms to reach their effect. Five mechanisms are described
(Chen and Mellman 2017).
1. Ligand blockade
Full length IgG therapeutic antibodies, antibody fragments (e.g., Certolizumab)
or receptor fusion proteins (Etanercept) prevent the ligand from engaging with
their cognate receptors.
2. Receptor blockade
Antibodies can be directed directly to their cognate receptors and thus inhibit
receptor activation. Examples are Tocilizumab, Natalizumab, Vedolizumab, and
Abatacept.
Fig. 20.3 Targets of monoclonal antibodies, fusion proteins and biosimilars in different disease
stages of immune-mediated diseases. Adapted from Burmester et al. (2014), Davis and Ballas
(2017), and Her and Kavanaugh (2016). Illustration: Oliver Hippmann, Schwarzenbruck
ADCC ADCP CDC (CDCC, CDCP)
C
314
ONCOGENIC PATHWAY BLOCKADE

Breast and metastatic colorectal cancer (EGFR), Sarcoma (PDGFR),
Neuroblastoma (GD2)
ONCOGENIC PATHWAY BLOCKADE
Makrophage Complement Trastuzumab
Lymphoma (CD20, CD30); Myoloma (CD38, SLAMF7) NK-cell
activation Cetuximab
FCR mAbs: Panitumumab
Ofatumumab Daratumomab C1q
mAbs: mAbs Pertuzumab
Obinutuzumab Brentuximab Elotuzumab C5a
Necitumumab Olaratumab Dinutuximab
Target/TAA: CD20 CD30 CD38, SLAMF7 TAA
TAA: EGFR (e.g. HER) PDGFR GD2
MAC
ANTIBODY-DRUG- Cytotoxic drugs,

CONJUGATES cytokines, drugs MODULATION OF IMMUNE RESPONSE
Metastatic melanom, non small cell lung carcinoma,
Breast cancer
urothelial carcinoma
ADI-Trastuzumab emtansine Isotope
HER2 Effector Memory Naive
TAA: HER2 Tumor CTL T-cell T-cell T-cell
Osteoclast-blockade
ANGIOGENESIS BLOCKADE
Metastatic colorectal/gastric cancer
Tumor Cytotoxic
Ranibizumab VEGF-R vascu- granules
Ramucirumab larization
Aflibercept VEGF
Bevacizumab
Zic-aflibercept
CD10 CD19 Hyperexhausted Exhausted
T-cell T-cell
OSTEOCLAST BLOCKADE Blinatumomab (nonrecoverable) (recoverable)
Bone metastases
Denosumab CD3 Checkpoint inhibitors
CAR CTLA-4: Ipivimumab PD-L1: Atezolizumab
RANKL T-cell PD-1: Nivolumab Durvalumab
RANK Pembrolizumab Avevolumab
(engineered)
RANKL
T-cells Osteoblast Osteoclast
bone stromal RETARGETING T-CELLS (CART) and BISPECIFIC ABS
cells ALL (CD10, CD19)
Bone Blinatumomab
T. Niehues
3. Receptor downregulation
Instead of blocking the receptor binding of the antibody might lead to a down-
regulation of receptors and to their internalization. Examples are Omalizumab,
Otelixizumab, Teplizumab, and Epratuzumab.
4. Binding to the cell surface receptor may result in depletion of all or a majority of
receptor carrying cells. Depletion of cells will be achieved by complement
mediated cytotoxicity (CMC) and/or antibody-dependent monocyte/macrophage
mediated phagocytosis ADCP (antibody dependent cellular phagocytosis) anti-
body-dependent cellular cytotoxicity (ADCC) (e.g., by NK-cells) (Fig. 20.2a).
Examples are Rituximab, Ofatumumab, Ocrelizumab, Alemtuzumab, Muromonab,
and Epratuzumab.
5. Signaling induction
Instead of blocking the receptor therapeutic antibodies may activate signals
after binding to the receptor. Examples are mAbs binding to the T-cell receptor
TCR/CD3, e.g. Teplizumab and Muromonab.
20.9 Indications and Efficacy
FDA-approved indications related to each agent are listed in Table 20.1.

Biologics (mAbs and fusion proteins) have been used in very different diseases
and disease subtypes. Biologics are used off label in many indications, which can-
not be covered in this chapter. There is strong evidence that they will benefit
patients in some situations which cannot easily be studied in clinical trials (e.g.,
Alemtuzumab (anti CD52) CAMPATH in stem cell transplantation used for condi-
tioning). In the following I will focus on main indication areas for biologics in the
treatment of immune mediated diseases and cancer. Cochrane and other meta-anal-
yses of the main indications (RA, Psoriasis, IBD, MS (Multiple Sclerosis)) were
reviewed.
The quality of meta-analyses can differ substantially. The type of funding
sources, author conflicts of interest, and author networks compromise study quality
and risk of bias as impressively shown for psoriasis (Gomez-Garcia et al. 2017;
Fig. 20.4 Targets in antibody-based cancer immunotherapy. Adapted from Hendricks et al.
(2017), Her and Cavanaugh (2016), Martin-Liberal et al. (2017), Weiner (2015), Batlevi et al.
(2016), and Sukshari et al. (2017) Chen (2017) Illustration: Oliver Hippmann, Schwarzenbruck.
ALL = acute lymphoblastic leukemia, CART = chimeric antigen receptor T-cells, CTL = cytotoxic
T-cell, CDCC = complement dependent cellular cytotoxicity, CDCP = complement dependent cell
mediated phagocytosis, MAC = membrane complex, TAA = tumor associated antigen, EGFR =
epithelial growth factor receptor, PDGFR = platelet derived growth factor receptor, GD2 = gluco-
lipidsialoganglyosid, PD1 = programmed cell death 1PD-L1 = programmed cell death ligand 1,
CTLA4 = cytotoxic T-lymphocyte associated protein 4, ADCC = antibody dependent cell medi-
ated cytotoxicity ADCP = antibody dependent cell mediated phagocytosis, VEGFR = vascular
endothelial growth factor receptor, VEGF = vascular endothelial growth factor, HER2 = human
epidermal growth factor receptor 2, RANK = receptor activator of nuclear factor kB, RANKL =
receptor activated of nf kB ligand
316 T. Niehues
Adverse effects
from wash-in
Responders Continue
Randomize trial drug
Screen Wash-in with Placebo
and trial drug (e.g. effect Withdraw
Patients
recruit etanercept) trial drug
with Study
polyarticular population
JIA
Nonresponders Excluded from trial
Fig. 20.5 Example of flawed trial design. The so-called withdrawal trial design is used in the
majority of controlled trials in polyarticular JIA. Flaws include false-high response rates by inclu-
sion of the placebo response and exclusion of non-responders as well as carry-over of adverse
events in both study (continued drug) and control (withdrawal drug) groups. Adapted from Niehues
(2015)
Sanz-Cabanillas et al. 2017). There clearly is an urgent need for investigator-initi-

ated trials which are independent are properly developed consensus treatment pro-
tocols. The majority of trials in all indication areas of therapeutic mAbs are
industry-initiated and industry-sponsored. A large body of data on therapeutic mAbs
derives from industry-sponsored registries with a high degree of bias. Patient regis-
tries are commonly used as a convenient shortcut to answer questions regarding
efficacy and safety of mAbs/fusion proteins to avoid expensive and laborious trials.
Registries have repeatedly shown to provide data of low and non-reliable quality
(Anderson 1994; Windeler 2014).
Trial design has been flawed in trials regarding industry-initiated clinical trials
on mAbs. A striking example are the many trials on mAbs in Pediatric Rheumatology:
With very few exceptions the so-called withdrawal trial design was used. The trial
design results in falsely high responder rates and lower rates of AEs in the study
drug and measures whether a drug can be withdrawn but not whether a drug is effi-
cacious (Niehues 2015) (Fig. 20.5). Any clinician is interested whether a drug pre-
scribed is effective and less so whether this drug can be withdrawn. Needless to say
that the drug wouldn’t be prescribed in the first place if it were not effective.
20.9.1 Immune Mediated Diseases
20.9.1.1 Rheumatoid Arthritis (RA)

In all of diseases below (RA, JIA, Uveitis, MS, IBD, etc.) information from trials is
of limited value because of the short observation periods in trials (max. 2 years) for
diseases that last for 30–40 years. Especially regarding safety data longer observa-
tion periods are key.
RA is characterized by acute and chronic inflammation in the synovium which is
associated with a destruction of joint tissues.
Early use of DMARDS (disease modifying antirheumatic drugs, e.g. Methotrexate
MTX) is key to the success of RA treatment (Burmester and Pope 2017; Moreland
2016). According to ACR and EULAR recommendations (ACR American College
of Rheumatology, EULAR European League against Rheumatism) initial treatment

does not consist of TNFα inhibitors but one DMARD or combination of two
DMARDs, non-steroidal and anti-inflammatory drugs (NSAIDS) and/or glucocorti-
coids (systemic and/or intra-articular). Only if there is no remission after 3–6 months
of treatment, a TNF inhibitor can be added. Meta-analyses showed that addition of a
biological to a conventional DMARD is more effective than a conventional DMARD
alone (Nam et al. 2017). In adults with RA who are naive to Methotrexate biologics
(Abatacept, Adalimumab, Etanercept, Golimumab, Infliximab, Rituximab) in com-
bination with MTX probably improve signs and symptoms of RA while the use of
TNF biologics alone does not make a difference regarding signs and symptoms of
RA or chances of RA remission (Singh et al. 2017).
20.9.1.2 Juvenile Idiopathic Arthritis (JIA)

Juvenile idiopathic arthritis usually has a much less severe disease course as com-
pared to RA and can be subdivided into Oligoarthritis and Polyarthritis (involving
more than five joints). Notably, there are no clinical trials on the use of biologics
in the most common subtype of JIA (persistent oligoarthritis). It is quite worrying
that a large proportion of children with JIA oligoarthritis are treated with biolog-
ics without any evidence from properly conducted clinical trials (Davies et al.
2017). In most JIA subtypes MTX has been shown to be effective repeatedly in
investigator-initiated trials. If Methotrexate fails, clinicians feel that TNF inhibi-
tors are efficient drugs, however, the evidence for the overall efficacy of biologi-
cals in JIA remains unclear as the trial design has been exclusively an inadequate
industry-initiated withdrawal design trial (see above, Fig. 20.5) (Niehues 2015).
In JIA with systemic onset (SJIA) (Stills disease) two randomized placebo-
controlled trials claimed efficacy for Tocilizumab and Canakinumab which were
industry-initiated and limited by their very short observation periods of their
double-blind phases (12 weeks, 29 days) (De Benedetti et al. 2012; Ruperto
et al. 2012).
20.9.1.3 Uveitis
Adalimumab can be considered as second-line immunomodulatory agent. In
patients with JIA associated uveitis and ongoing ocular inflammation despite sys-
temic and local glucocorticoid therapy Adalimumab has been shown to be effective
in an investigator-initiated trial (Ramanan et al. 2017).
Data on non-JIA associated inflammatory uveitis and biologicals are lacking. In
Behçet’s disease and its ocular manifestations Infliximab and Adalimumab can be
considered as first-line treatment. Infliximab and Adalimumab are considered as
potential second-line immunomodulatory agents for Posterior Uveitis, Panuveitis,
severe Uveitis associated with seronegative spondylarthropathy, Scleritis. In con-
trast, Etanercept has been linked to the induction of De novo-Uveitis.
20.9.1.4 Systemic Lupus Erythematosus (SLE)

SLE is characterized by high titers of antinuclear antibodies, in particular anti-dou-
ble-stranded DNA antibodies which are thought to play a role in SLE pathogenesis.
Therefore, B-cells and antibody-producing plasma cells have been thought to be an
318 T. Niehues
important target for biologics in the disease, but the results of some well-designed
controlled trials (e.g., rituximab) were disappointing (Merrill et al. 2010; Rovin
et al. 2012).
20.9.1.5 Psoriasis
Psoriasis is a chronic inflammatory skin disease (Feldman 2017). Limited plaque
psoriasis responds to topical corticosteroids and emollients, or alternatively Vitamin
D analogs or topical retinoids. Severe psoriasis requires phototherapy or systemic
therapy with MTX, cyclosporine (CSA), or biologics (anti-TNF agents, anti-IL12/
IL23, anti-IL17) (see Tables 20.1 and 20.2). The British dermatologists recommend
offering biologics to patients who require systemic therapy, if MTX and CSA have
failed, are not tolerated or contraindicated, and psoriasis is extensive or particularly
severe at localized sites (Smith et al. 2017). Biologics can be considered earlier and
after MTX has failed, if there is psoriatic arthritis.
20.9.1.6 Multiple Sclerosis (MS)

MS is an immune mediated inflammatory demyelinating disease of the central ner-
vous system that is a leading cause of disability in young adults. Biologics (cyto-
kines: Interferon β1a¸mAbs: Natalizumab, Daclizumab, Alemtuzumab) play an
important role in the treatment of patients with relapsing remitting multiple sclerosis.
The initial choice depends on disease activity and patient values and preferences
(Olek 2017). For prevention of disability only Natalizumab shows a beneficial effect.
Natalizumab is recommended for patients with more active disease and patients who
value effectiveness above safety and convenience. Daclizumab, a humanized mono-
clonal antibody binding the alpha chain of the high affinity IL2 receptor and
Alemtuzumab depleting CD52 positive cells (T-cells, B-cells) have both been shown
to reduce disability progression compared to placebo, however, their clinical utility
is likely to be limited by severe adverse events (cytokine storm, serious infections,
autoimmune disorders). Rituximab and Ocrelizumab (licensed in 2017) targeting
CD20 appear to be effective but long-term data from trials are lacking.
20.9.1.7 Inflammatory Bowel Disease

In mild or moderately active Crohn’s disease immunomodulators or biologics
are not necessary. In patients who are refractory to treatment with first-line
agents like glucocorticoids, 5-aminosalycilate therapy and antibiotics
Azathioprine, Methotrexate, and/or mAbs are used (TNF/Integrin inhibitors)
(see Table 20.1).
In a recent systematic review on induction and maintenance of mucosal healing
in moderate to severe Crohn’s disease or ulcerative colitis (trial observation peri-
ods of 6–12 weeks for induction, 32–54 weeks for maintenance trials) anti-TNF
treatment was more effective than placebo for maintaining mucosal healing in
Crohn’s disease. In ulcerative colitis, anti-TNFs and anti-integrin therapy with
Vedolizumab were more effective than placebo and Adalimumab was inferior to
Infliximab. Both Infliximab and Adalimumab were similar in Crohn’s disease
(Cholapranee et al. 2017).
20.10 Adverse Events (AEs)
Target-related and unrelated AEs for biologics are listed in Table 20.2. In general
terms, pharmacodynamically large quantities (hundreds of milligrams per milli-
liter) have to be administered to achieve target saturation and the half-life of IgG
is usually quite long at around 3–4 weeks. Adverse events may occur Iate and
span over a longer time-span than in conventional chemical compound drugs.
AEs tend to be underreported. A striking example is the TENDER trial in
sJIA. Although published in a top-ranking journal, fatal adverse events in the study
drug group are not mentioned in the abstract (De Benedetti et al. 2012):
Six patients of 112 included discontinued treatment (increase in liver enzymes,
macrophage activation syndrome). Three patients died during treatment from pneu-
mothorax at week 50, a traffic accident in week 90 and a streptococcal sepsis at
week 104. Three more patients in the treatment group died after being withdrawn
from the study, two died from pulmonary hypertension after 6 months while one
more patient died from probable macrophage activation syndrome 13 months after
withdrawing from the study.
Long-term pharmacosurveillance is key in the safety of these many new com-
pounds. Pharmacosurveillance within the biological drug class is almost exclusively
done in industry-financed patient registries. Patient registries have multiple flaws
and are not the adequate tool to monitor long-term side-effects of mAbs (see above).
Mechanistically, AEs can be classified into the categories immune stimulation,
immunodeficiency alteration of homeostasis, off target activity (Boyman et al.
2014; Davis and Ballas 2017).
20.10.1 Immune Stimulation
Standard infusion reactions (SIRs) occur but they usually remain moderate (Boyman
et al. 2014). Therapeutic mAbs resemble physiological antibodies and are highly
complex molecules. They are not identical to the human proteins and thus are rec-
ognized by the hosts’ immune system leading to hypersensitivity reactions (Picard
and Galvao 2017). Immediate hypersensitivity reactions are the result of degranula-
tion of mast cells, may be more severe, and can progress to life-threatening condi-
tion within minutes. Immediate hypersensitivity reactions usually do not occur after
the first infusion and are triggered by subsequent infusions similar to IgE mediated
reactions. Apart from neutralizing the biologic agent anti-drug antibodies can cause
clinical hypersensitivity reactions.
Antibodies to a target can lead to activation of the receptor-bearing cell (e.g.,
T-cell). This has been documented for Muromonab leading to a cytokine storm and
severe hypotension and multi-organ failure, etc. The most drastic example of a cyto-
kine storm and uncontrolled immunostimulation occurred when an anti-CD28 acti-
vating monoclonal antibody TGNIH2 was infused resulting in a cytokine storm and
multiorgan dysfunction (Suntharalingam et al. 2006) which received world-wide
press coverage.
320 T. Niehues
20.10.2 Immunodeficiency, Infections and Cancer
Efficient targeting of an important molecule in immunophysiology may lead to a

clinical phenotype resembling an inborn Primary Immunodeficiency. Similarly,
autoimmune diseases in which endogenous monoclonal autoantibodies are pro-
duced against cytokines (e.g., IL17) lead to the clinical phenotype of chronic muco-
cutaneous candidiasis that is indistinguishable from inherited primary
immunodeficiency (caused by mutations in genes responsible for development of
TH17 cells: STAT1 gain of function). Therefore, most of the infections occurring in
patients treated with exogenous mAbs against well-known immune system targets
show characteristic infectious disease profiles, e.g. tuberculosis reactivation upon
anti-TNF treatment (Fig. 20.6).
Fig. 20.6 Tuberculosis
reactivation after 7 months
of adalimumab treatment in
a 16-year-old boy with
Crohn’s disease. MRI shows
interstitial changes, enlarged
lymph nodes, infracarinal
tuberculoma with colliqua-
tive necrosis
Fig. 20.7 Multiple squamous cell carcinomas on lower limb apparent after commencing
ustekinumab as treatment of moderate to severe plaque type psoriasis. Also note pre–existing
actinic damage and evidence of chronic venous stasis. Adapted from Young and Czarnecki (2012)
Moreover, it is known that in primary immunodeficiencies immunosurveillance

is hampered and thus cancers may arise (e.g., squamous cell carcinoma arising upon
anti-IL-12/23 treatment, Fig. 20.7). Major indications for biologic treatments
(mAbs, biosimilars, and fusion proteins) are immune mediated diseases and cancer.
Both immune-mediated diseases and/or cancer cause a disease-related immunodefi-
ciency. The use of immune-modulating agents and biologics amplifies this immuno-
deficiency in patients. The combination of both, a conventional immunosuppressive
drug and a biological drug in the same patient significantly increases the risk of
infection and/or cancer. So outside of controlled trials it is difficult to clearly allo-
cate severe side-effects to biologics only.
20.10.3 Alteration of Homeostasis and Inflammatory Diseases
Some of the targets blocked by therapeutic mAbs may be important in maintaining

peripheral tolerance, e.g. cytokines important for both pro-inflammatory and anti-
inflammatory functions. Accordingly blocking TNF may lead to an array of autoim-
mune diseases, e.g. Uveitis (Fig. 20.8) vasculitis, demyelinating CNS disease
(Figs. 20.9 and 20.10), IL17 blockade (Secukinumab) may lead to Crohn’s disease.
A large array of autoimmune diseases, so-called immune-related adverse events
(IrAEs) have resulted from blocking molecules important for T-cell activation
(checkpoint inhibitors), some of them fatal.
322 T. Niehues
Fig. 20.8 Induction of

uveitis by etanercept.
Funduscopy of left eye
showing snow-balls in the
inferior vitreous cavity
(Fonollosa A, Artaraz J, Les
I, Martinez-Berriotxoa A,
Izquierdo JP, Lopez AS,
Gardeazaba J, Berasategui
B, Martinez-Alday N:
Sarcoid intermediate uveitis
following etanercept
treatment: a case report and
review of the literature. Ocul
Immunol Inflamm
2012,20(1):44–48)
Fig. 20.9 Cutaneous AEs of TNF blocking agents: Verrucae vulgares after 3 months treatment
with adalimumab in a 13-year-old girl with polyarticular JIA (lower panel); vasculitis on right calf
of 13-year-old girl treated with etanercept for polyarticular JIA (upper panel)
Fig. 20.10 Demyelinating CNS disease 57-year-old man with RA managed with infliximab, metho-
trexate, and low-dose prednisone presented with 5 days of progressive encephalopathy. Brain MRI
with and without contrast. Fluid-attenuated inversion recovery sequences demonstrate T2 hyperin-
tense lesions in the pons, right middle cerebellar, bilateral basal ganglia, left frontal and parietal lobes
(not shown). The patient had started infliximab 4 months prior to presentation. The patient’s neuro-
logic examination and the brain MRI lesions progressed over several days, and he was treated with
high-dose steroids for a presumed autoimmune demyelinating syndrome. The patient died from septic
shock. There was pathologic proof of a demyelinating process following TNF antagonism: Brain his-
topathology demonstrated an acute demyelinating process. Adapted from Bradshaw et al. (2016)
20.10.4 O
ff-Target Activity (e.g. Progressive Multifocal
Leukoencephalopathy PML)
As antibodies are highly specific for their target it appears unlikely that off-target
activity occurs. However, regarding severity and localization some unexpected
clinical findings have been made upon administration of biologics (e.g., CNS
side-effects: PML in Integrin inhibitors, TNF inhibitor side-effects in the skin).
324 T. Niehues
20.11 Summary
Immunotherapy with antibodies has moved from the use of polyclonal IgG plasma
derived mixtures to engineered, monoclonal abs. Precise understanding of the
protein-chemistry of antibodies, and modern production technologies allow us to
generate designer abs, that almost resemble human abs. They target specifically
molecules that appear central to pathophysiological processes. While this has been
a significant medical progress, caution is strongly advised:
1. These new drugs need to be as rigorously tested for efficacy and safety as con-
ventional drugs within properly designed, randomized, and placebo-controlled
clinical trials and followed up in independent registries long-term. Notably in
many biologics this has not yet been done. Just because these drugs are highly
complex and very precise in targeting it doesn’t mean they are effective and safe.
2. Nihil nocere is a prime duty for every ethically aware clinician. MAbs, biosimi-
lars, and fusion proteins have repeatedly caused severe AEs and death, which to
some degree appears to be underreported. In light of their potential harm and
currently very high cost for our health systems a strictly evidence-based approach
for using biologics is needed.
Acknowledgement Without the careful and skillful help of Andrea Groth the preparation of this
manuscript would not have been possible. I thank Kathrin Siepermann, Gregor Dückers, and Antje
Ballauff for discussing interesting cases of treatment with biologics within our team at the HELIOS
Children’s Hospital in Krefeld, Germany. I thank Ina Bruins for her additional help in manuscript
preparation.
Table 20.11 Target-and target system associated adverse events of therapeutic monoclonal antibodies, biosimilars and fusion proteins (licensed by FDA, some
taken from market, some in development): Hematopoietic cells (granulocytes, platelets)
Target Brand
system Target molecule Antibody name Physiology/pathophysiology (Fig. 3)
Granulocytes (adhesion molecules)
Alpha-4 integrin Natalizumab Tysabri A first step in migration of inflammatory cells (especially leukocytes) to the inflamed tissue is
their adherence to endothelial cells in vessels. The endothelial cells express selectins and
integrins. Adhesion molecules are expressed both on the endothelial side and the surface of
inflammation cells (neutrophil granulocytes). Inability of neutrophil granulocytes to adhere to
endothelial cells or blood vessels in inborn adhesion defects leads to very severe necrotic
infections with a lack of pus and extremely high leukocyte numbers in blood. Neutrophil
granulocytes that adhere to endothelial cells express Beta integrins (CD18, LFA1 lymphocyte
function associated antigen) on the cell surface of neutrophil granulocytes. These integrins bind
to adhesion molecule receptors on activated endothelial cells (ICAM1, ICAM2, MadCAM-1).
Natalizumab binds to the α4 subunit found in α4β1 (VLA-4) and α4β7 integrins which are
ligands for VCAM 1 and MADCAM 1 adhesion molecules.
Alpha-4 integrin Vedolizumab Entyvio
CD11a Efalizumab Raptiva
Adverse Events (AEs):
Blocking the ingress of neutrophils into inflamed tissue sites may lead to immunodeficiency and severe infections: localized herpes zoster reactivation,
pneumonia, urinary tract infections.
PML progressive multifocal leukoencephalopathy results from reactivation of the JC virus (John Cunningham) and has been fatal in many cases. Inhibition
of entry of CD4 cells to the CNS as well as a mobilization of stem cells that might harbor the JC virus are hypothesized to be responsible for this very
severe side-effect. Natalizumab as well as Efalizumab had been withdrawn from the market but in need of effective drugs for multiple sclerosis
Natalizumab was reintroduced in 2006. The risk for JC virus reactivation depends on the length of treatment and prior use of immunosuppressants. Because
of the PML risk Natalizumab should not be combined with other immunosuppressants.
Platelets
GPIIb/IIIa Abciximab ReoPro GPIIb/IIIa is expressed on activated platelets
Adverse Events (AEs)
Excessive or abnormal bleeding sometimes associated with thrombocytopenia.

Hypersensitivity reaction including anaphylaxis
325
Table 20.12 Target-and target system associated adverse events of therapeutic monoclonal antibodies, biosimilars, and fusion proteins (licensed by FDA,
326
some taken from market, some in development): Lymphocytes, T-Lymphocytes, T-Lymphocyte/APC (synapse)
Target Target
system molecule Antibody Brand name Physiology/pathophysiology) (Fig. 4)
Lymphocyte subpopulation
CD52 Alemtuzumab Campath, Lemtrada CD52: cell surface protein of unknown function, found on neutrophils,
monocytes, macrophages, lymphocytes including T, B, and NK cells
Adverse events (AEs):
It may induce severe lymphopenia, sometimes for more than 10 years. Phenotype similar to severe combined immunodeficiency. Severe systemic viral,
fungal, bacterial, and protozoal infections have been described as well as TB reactivation. Viruses (VZV, HSV, CMV, EBV) fungi (Candida, aspergillus,
cryptococcus, mucor). Due to altered hematopoiesis ITP hemolytic anemia neutropenia and pancytopenia have been observed. Live vaccines should not be
used. Emergence of autoimmune thyroid disease and immune thrombocytopenia with a case of fatal intracranial hemorrhage has been documented.
T-lymphocytes
IL2RA Basiliximab Simulect IL-2, the most important cytokine for T-cell proliferation and activation, binds to
IL2R Daclizumab Zinbryta (Zanaprax until the IL2 receptor. The IL2 receptor is composed of the alpha chain and a common
2009) gamma chain which is a shared receptor for other cytokines. Recombinant human
Denileukin diftitox ONTAK IL2 (Aldesleukin) which may be useful in the activation of tumor specific
lymphocytes (metastatic renal cell carcinoma, metastatic melanoma)
CD3 Blinatumomab Blincyto CD3: classical lineage specific marker for T-cells. By stimulating CD3,
Muromonab Orthoclone OKT3 Teplizumab is supposed to induce IL10-producing CD4 cells and CD8 positive
Teplizumab N. N. T-regulatory cells (Tregs)
CD2 Alefacept Amevive CD2: Expressed on T cell, B cells, and NK cells.
By activating the IL2 receptor T-cells may be overtly stimulated leading to post administration anaphylaxis and a so-called cytokine storm, which may be
fatal. Hypotension, angina pectoris, arrhythmia, tachycardia, dyspnea, pulmonary edema, thrombocytopenia, thrombosis all have been described.
Basiliximab is used in transplant patients thus underlying diseases or coadministered drugs may also be responsible for AEs. Infections observed with IL2
blocking agents have been respiratory tract infections, herpes simplex, and herpes zoster skin infections.
Daclizumab was taken from the market in 2009 and later reintroduced for MS patients. Muromonab (Orthoclone OKT3) was the first licensed therapeutic
monoclonal antibody and used in renal and liver transplantation but was withdrawn from the market in 2010. It was effective in preventing graft rejection
but caused frequent systemic infections as well as cytokine storm. Teplizumab is not expected to have the same adverse events as muromonab because it is
engineered not to bind to FcR
T. Niehues
The LFA3 IgG1 FC fusion protein (LFA3 is the ligand for CD2), alefacept was used in psoriasis but withdrawn from the market in 2011.
T-lymphocyte/APC (synapse)
B7-1 Abatacept Orencia As the crucial initial step of T-cell activation, the immunological synapse is
(CD80), formed between antigen-presenting cell (APC) and T-cell. Within the synapse
B7-2 there are many targets for the use of biologics (Figure 3). Following ligand
(CD86) receptor pairs can be inhibited by mabs: CD28/CTLA4 and CD80/CD86, PD1
CD80, Belatacept Nulojix and PD-L1/PD-L2 (CD274)CD27 and CD70, CD40 ligand and CD40, CD137,
CD86 OX40, LAG3, TIM3 may be future targets.
CTLA-4 Ipilimumab Yervoy
PD-1 Nivolumab Opdivo
Pembrolizumab Keytruda
PD-L1 Atezolizumab Tecentriq
Durvalumab Imfinzi
Avelumab Bavencio
Immune checkpoint inhibitors have a unique and broad profile of adverse events (immune related adverse events irAEs), expected to occur in about
10–20% of cases. Temporary immunosuppression with other immunosuppressive drugs may be necessary (e.g., with corticosteroids). (Postow, Journal of
clinical oncology 2015;33:1974). Fatal Adverse Events (FAEs) are significantly increased with ipilimumab. (Zhu, Exp Opin Drug Saf 2017; Zhang, Eur J
Cancer 2017). Frequently a reticular macropapular erythematous rash on extremities or trunk has been reported as well as Stevens Johnson Syndrome and
Toxic epidermal necrolysis. Diarrhea results of colitis and occurs approximately 6 weeks into treatment as a result of altered homeostasis.
Endocrinopathies effecting hypopituitary, adrenal, and thyroid glands occur. Opportunistic infections result from treating the irAEs. Autoimmune
encephalitis due to NMDA receptor antibodies has been described.
327
Table 20.13 Target-and target system associated adverse events of therapeutic monoclonal antibodies, biosimilars and fusion proteins (licensed by FDA, some
328
taken from market, some in development): B-cells and B-cell specific cytokines
Target molecule Antibody Brand name Physiology/pathophysiology (Fig. 3)
BLyS (BAFF) Belimumab Benlysta B-cell activating factors are BAFF (B-cell activating factor, synonym: BLYS and APRIL (a
proliferation inducing ligand), and share the ability to bind to TACI (= Transmembrane
activator and CAML interactor) expressed on mature B-cells, plasma cells, and activated
T-cells and BCMA (also known as B-cell maturation antigen) expressed on B-cells and
plasma cells. BAFF can also interact with BAFF receptor
Monoclonal antibodies can interact with B-cell differentiation by binding to BAFF and
APRIL (Atacicept = fusion protein of human IgG FC protein and TACI); Belimumab or
Tabalumab, block BAFF
CD20 Ocrelizumab Ocrevus CD20 is not expressed on pro B-cells or antibody-producing plasma cells, an exclusive
marker for mature B-cells and expressed on more than 95% of B-cells in blood and lymphoid
organs
Rituximab Rituxan
Tositumomab Bexxar
CD22 Epratuzumab Lyphocide CD22 is a member of the lectin-like immunoglobulin superfamily and expressed to a higher
(planned name) extent in naïve versus memory B-cells. It is part of the CD19/CD21/CD22/BCR (B-cell
receptor complex)
Belimumab seems to have a less broad adverse event profile than the B-cell depleting antibodies. However, in clinical trials cases of opportunistic
infections with acinetobacterand CMV were seen. The general experience is still limited, fatal infections have been described.
Rituximab has been widely used, a lot of side-effects have been reported, many of them occur in conjunction with chemotherapy/steroids for Lymphoma.
Regarding infections one would expect the clinical phenotype of a primary antibody or B-cell deficiency (like in Bruton’s agammaglobulinemia).
Accordingly, bacterial infections like osteomyelitis, otitis, conjunctivitis, cellulitis all have been observed. Accordingly, PJP (Pneumocystis jirovecii
pneumonia) with poor outcome (30% fatal) (Martin-Garrido, Chest 2013) and PML have been observed. A recent study found an increased incidence of
AML (acute myeloid leukemia) after Rituximab treatment for B-cell lymphomas. (Tao, British Journal of Hematology 2017).
Epratuzumab is used in SLE systemic lupus erythematosus and leads to a moderate peripheral B-cell reduction (approximately 30%) and inhibition of
B-cell proliferation.
T. Niehues
some taken from market, some in development): TNF
Target molecule Antibody Brand name Physiology/pathophysiology (Fig. 3)
TNF Adalimumab Humira Cytokines are small proteins
TNF Certolizumab pegol Cimzia (5–20 kilodalton) that are important
TNFα and TNFβ Etanercept Enbrel in cell signaling. They are part of a
complex network regulating humoral
TNF Infliximab Remicade
and cell-based immune responses.
TNF Infliximab-dyyb Inflectra TNF is involved in cytokine and
TNF Golimumab Simponi Aria chemokine inducing apoptosis,
TNF Golimumab Simponi endothelial cell activation, induction
TNF Adalimumab-atto Amjevita of adhesion molecules and growth of
TNF Infliximab-abda Renflexis new blood vessels as well as
regulation of fibroblasts,
synoviocytes, and osteoclasts.
The most characteristic side-effect is reactivation of latent tuberculosis (see Fig. 7). Higher incidence of pneumonia and sepsis as well as serious bacterial
(particularly intracellular) and viral infections. Opportunistic infections such as histoplasmosis, PJP, candida, and aspergillus have been described, in
many of the reported cases concomitant immunosuppressive medication was present
Immunologically mediated side-effects include fatal infusion reactions, reactivation of hepatitis B and cytopenias. Outside of the immune system cardiac
arrhythmias and bowel obstruction have been described: Caution has to be used in patients with Congestive Heart Failure. A whole array of autoimmune
diseases or immune mediated diseases associated with TNFα blocking therapy have been described: cutaneous side effects such as psoriasis, cutaneous
lupus, necrotizing vasculitis, induction of anti double-stranded DNA antibodies, dermatomyositis, polymyositis, antiphospholipid antibody syndrome,
exacerbations of IBD have been described. Uveitis has been repeatedly induced by Etanercept (see Fig. 6), in 2007 Ramos Casals had already described
234 cases of autoimmune diseases induced by TNF target therapies (Ramos Casals, Medicine 2007;86:242)
It is controversial to what extent anti-TNF treatment is associated with the induction of lymphomas (Hodgkins, Cutaneous T-cell Lymphoma). Rare but
almost uniformly fatal is hepatosplenic γδ T-cell-lymphoma in IBD patients. There is an increased risk of melanoma and non-melanoma skin cancers.
Regular skin cancer screening is advised. In many meta-analyses there has been no significant increase in the occurrence of lymphomas. It is difficult to
discriminate between the effects of the underlying disease, concomitant medication and induction by TNF antagonists
CNS leukoencephalopathy and cerebral edema have been described with Etanercept treatment (Deutsches Ärzteblatt 2015;40:b1357). Demyelinating acute
disseminated encephalomyelitis (Fig. 10) has been described as well as peripheral neuropathies (Guillain Barré syndrome), multifocal motor neuropathy,
chronic inflammatory demyelinating polyradiculoneuropathy. TNF inhibitors should not be given in first degree relatives of patients with Multiple Sclerosis
329
330
some taken from market, some in development): Interleukin 1 and Interleukin 6 w

Target Brand
system Target molecule Antibody name Physiology/pathophysiology
Interleukin 1
Interleukin-1 type Anakinra Kineret IL1 has pleiotropic effects: It initiates inflammation and fever, as one of the most important
I receptor (IL-1RI) effects. IL1α and β are important for infection immunity. Uncontrolled release of
IL1B Canakinumab Ilaris Interleukin1β leads to severe tissue damage and systemic inflammation (deafness and
IL-1 beta (IL-1β) Rilonacept Arcalyst blindness due to optical and acoustic nerve inflammation, treatment-resistant fever, joint
destruction, etc). IL1 receptor antagonist (IL-1RA) is a soluble, naturally occurring
antagonist which was the model for the development of the synthetic IL-1RA Anakinra.
Bacterial and viral infections (with a higher rate in Canakinumab due to its long half-life) opportunistic infections appear not to be significantly increased.
The FDA has not approved Canakinumab for the treatment of gout because of increased incidents of severe infections particularly in the elderly
population. (Dinarello, Nature reviews Rheumatology 2016;12:). Fatal myocarditis has been described in a child with systemic onset juvenile idiopathic
arthritis on treatment with Anakinra. (Zeft, Pediatric Rheumatology 2012;10:8). Immune mediated side-effects of all IL1 antagonists include pneumonitis,
colitis, hepatitis, endocrinopathies, nephritis, dermatitis, encephalitis, psoriasis, and vitiligo. In some cases neutropenia occurs
T. Niehues
Interleukin 6
IL6 Siltuximab Sylvant IL6 is a pro-inflammatory cytokine and binds to the IL6 receptor composed of 2 functional
IL6R Tocilizumab Actemra chains IL6 receptor α and a common signal transducing chain (GP130). IL6 receptor α is
expressed only on a few cell types (mainly hepatocytes, leukocytes). However IL6 is also
able to activate GP130 expressing cells which are abundant. IL6 induces the acute phase
proteins in the liver (e.g., C-reactive protein), is part of inducing fever and leukocytosis,
induces hematopoiesis and angiogenesis at inflammation sites. Increased IL6 in chronic
inflammation induces anemia through induction of hepcidin from hepatocytes, activates
T- and B-lymphocytes, plasma cells and induces the differentiation of T-helper 17
lymphocytes (among other cytokines IL1β, TGFβ, IL21, IL23). It induces ACTH, cortisol,
and prolactin and inhibits the secretion of TSH and growth hormones (in chronic
inflammation: growth retarding). IL6 increases insulin resistance and is associated with the
development of a type-2 diabetes mellitus. It induces the cytokine RANKL and may
induce osteoporosis. Il-6 mediated fibroblast proliferation may result in joint destruction.
Blocking of IL6 may have quite drastic adverse events with a relatively high number of deaths in trials. Blocking IL6 effectively means that there will be
no c-reactive protein and ESR elevation, which normally can be used as lab tests to detect inflammation. Severe infections have been reported: Pneumonia,
urinary tract infection, gastroenteritis, diverticulitis, bacterial arthritis, and sepsis. Infections include opportunistic infections with aspergillus, candida,
and pneumocystis species. Therapy should not be started in patients with active infections of any kind. Live viral vaccines should not be given. The risk of
acquiring a serious infection is > 10% on Tocilizumab trials. Macrophage activation syndrome is common and fatal in some cases. Other fatalities in
Tocilizumab trials have been due to bacterial sepsis, Pneumothorax, Pulmonary hypertension, vasculitis, and heart failure (reviewed in Machado 2017).
Infections with hepatitis B or C may severely reactivate. Neutropenia (< 1000 neutrophils/μL) occurs in some patients
In patients with systemic onset juvenile idiopathic arthritis (sJIA) one anaphylactic reaction was observed as well as a gastrointestinal hemorrhage from
diffuse acute or chronic colon ulceration. Liver enzyme and LDL cholesterol elevation was observed
Regarding immune mediated adverse events the occurrence of psoriasis or the worsening of a pre-existing psoriasis has been reported (Deutsches
Ärzteblatt 111:14, April 2014 b25)
331
332
some taken from market, some in development): Interleukin 4 and Interleukin 5

Target system Target molecule Antibody Brand name Physiology/pathophysiology (Fig. 3)
Interleukin 4
IL4RA Dupilumab Dupixent IL4 is the signature cytokine for TH2 cells.TH2 cells stimulate antibody
production by B-cells and augment eosinophil responses. TH2 cells produce
IL4, IL5, IL10, IL13. Th2 cells carry IL4 receptors and the cytokines IL5
and IL13 are key to IgE production. Increased activation of TH2 cells is
characteristic of allergic diseases.
Dupilumab has been approved by the FDA just recently, there is very limited experience on the safety of this drug. No serious adverse events were
reported in the trial on persistent asthma with elevated eosinophil levels (Wenzel, NEJM 2013;368:2455)
Interleukin 5
IL5 Mepolizumab Nucala
Reslizumab Cinqair
Reactions at the site of injection have been reported more often than in placebo mepolizumab)
For reslizumab oropharyngeal pain has been the most common adverse effect. The experience with these drugs is still very limited
T. Niehues
some taken from market, some in development), Interleukin 17, Interleukin 12 and Interleukin 23, GM-CSF
Target Target Brand
system molecule Antibody name Physiology/pathophysiology
Interleukin 17
IL17A Ixekizumab Taltz IL17 has a central role in the defense against extracellular bacteria and fungi. IL17 is the signature
IL17A Secukinumab Cosentyx cytokine for TH17 cells, which respond to IL6 and TGFβ and IL23, IL17. IL17 is a highly
IL17RA Brodalumab Siliq inflammatory cytokine acting on immune cells, epithelial cells, and fibroblasts. IL17A and IL17F are
specifically upregulated in inflammatory conditions leading to recruitment and activation of
neutrophils, lymphocytes, and macrophages and tissue damage, including the expression of matrix
metalloproteinases. TH17 cells are thought to be the main culprit in the initiation and progression of
chronic inflammatory disease (Review Burmester, Nature Reviews Rheumatology 2014; 10:77–87).
Effector functions of TH17 cells also appear to be osteoclastogenesis and bone erosion
Severe infections with extracellular bacteria and fungi and worsening symptoms in patients with Crohn’s disease (a reduced control of candida in the gut
microbiome could be a reason). For Brodalumab reports of increased suicidal ideation were unexpectedly observed, which may be due to an increase of
IL17 concentration in the periphery when IL17 receptor is blocked. (Dinarello, Nature Reviews rheumatology 2016;12)
Interleukin 12, Interleukin 23
IL12, IL23 Ustekinumab Stelara IL12 and IL23 are members of a cytokine family that regulate early-phase immune responses
towards activation of TH1 and TH17 cells. IL12 and IL23 play a key role in the psoriatic plaque and
mediate inflammation. IL12 and IL23 share a peptide chain (P40) that is targeted by Ustekinumab.
Pure IL23 inhibitors (anti P19) Tildrakizumab and Guselkumab are being tested.
GMCSF/GMCSF-R
GMCSF-R Mavrilimumab N. N. GMCSF is involved in the generation, survival and activation of cells from the myeloid
common β compartment. It regulates the function of neutrophils, eosinophils, and macrophages within the
chain proinflammatory network. It acts through the GMCSF receptor α heterodimer of an alpha chain for
specific binding and a common β-chain shared with IL3 and IL5 receptors
Disseminated infection caused by mycobacteria, salmonella, and BCG was an expected finding (Molinelli, Current drug safety 2016;11:35). Common AEs
are respiratory tract infections, diverticulitis, cellulitis, pneumonia, appendicitis, sepsis, osteomyelitis, cholecystitis, gastroenteritis, bronchitis, and urinary
tract infections. Non-melanoma skin cancer has been described (Figure 8) (Ehrmann, Inflammatory bowel disease 2012;18:e199; Young, Australian
journal of dermatology 2012;53:57–60.) In the latter report multiple squamous cell carcinomas developed 2–5 months after starting Ustekinumab. A single
333
case of posterior leukoencephalopathy syndrome is reported (Gratton, Acta dermatologica 2011;147:1197)

Pulmonary alveolar proteinosis can be caused by endogenous autoantibodies against GMCSF, so this may be a safety concern with mavrilimumab
334
some taken from market, some in development): complement, vessels and vascular growth, bone (osteoclast differentiation)
Target system Target molecule Antibody Brand name Physiology/pathophysiology
Complement
Complement Eculizumab Soliris The complement protein cascade is key to the recognition and killing of encapsulated
component 5 bacteria and clearance of immune complexes from blood. Dysregulation of
complement activation, e.g. unregulated production of C5A may result in hemolytic-
uremic syndrome (HUS)
Expectedly, a blockade of C5 will mimic inherited complement deficiency. Inherited deficiencies of complement are associated with susceptibility to
infections with Neisseria species. Neisseria meningitidis is a significant risk, upon eculizumab treatment. Encapsulated bacteria of concern are
Streptococcus pneumoniae, haemophilus, gonococci (Gleesing, Pediatric infectious disease journal 2012;31:543)
Vessels Vascular growth
VEGF, PIGF Aflibercept Eylea VEGF, PIGF1 and 2 are key to angiogenesis, which is a key feature of new
VEGF Bevacizumab Avastin inflammation, macular edema, or tumor spread and tumor growth
VEGFR1 Ranibizumab Lucentis
VEGFR2 Ramucirumab Cyramza
VEGF, PIGR Ziv-aflibercept Zaltrap
Problems with wound dehiscence can lead to the development of gastrointestinal perforation sometimes associated with intraabdominal abscess
Bleeding can be serious. Intracranial hemorrhage has been reported (Nishimura, World Journal of Gastroenterology 2011;17:4440). Fatal Hemoptysis has
occurred quite frequently in patients with lung cancer. Hypertensive crisis, nephrotic syndrome, congestive heart failure are listed in the FDA warnings.
Aflibercept and Ranibizumab are administered intravitreally into the eye and major safety concerns include endophthalmitis, retinal detachment, increased
intraocular pressure. Arterial thrombotic events such as strokes or myocardial infarctions have been observed.
In Ziv-aflibercept perforation, gastrointestinal hemorrhage and reversible posterior leukoencephalopathy have been described, notably in combination
with 5-FU and irinotecan based chemotherapy.
T. Niehues
Bone
RANKL Denosumab Prolia, Tumor cells interact with bone matrix and provoke osteoclast activation and bone
Xgeva destruction. Osteoclasts are activated by receptor activator of NFκb ligand (RANKL).
RANKL binds to receptor activator of nuclear factor kappaB (RANK). RANK
(CD265) is found on pre-osteoclasts. Bone homeostasis relies on constant and
continuous balance between bone breakdown by osteoclasts and bone synthesis by
osteoblasts. The absence of RANKL-RANK signaling prevents to some extent bone
resorption/destruction. Breast and prostate cancer as well as some hematological
conditions like multiple myeloma metastasize to the bone
Denosumab is contraindicated in hypocalcemia. Both calcium and vitamin D supplementation need to be completed before start of Denosumab therapy.
As RANKL is also expressed on some T-cells immune mediated side effects may occur, a slightly increased number of serious infections (mainly
bacterial) have been observed (Watts, Osteoporosis international 2012;23:327)
335
Table 20.19 Target-and target system associated adverse events of therapeutic monoclonal antibodies: Epithelial growth factor receptors and other tumor
336
associated antigens (TAA)

Target molecule Antibody Brand name Physiology/pathophysiology
HER2 Ado-trastuzumab Kadcyla The epithelial growth factor receptor EGFR acts as a point of integration for signals arising
emtansine from g-protein-coupled receptors and cytokine receptors and is also activated by
EGFR Cetuximab Erbitux neurotransmitters, lymphokines, and stress inducers. The epithelial growth factor receptor
signaling is key to the growth and development of human carcinomas. (Review Chiavenna
SM, Journal of Biomedical Science 2017. 24:15)
GD2 Dinutuximab Unituxin Glykolipiddisialoganglioside (GD2) belongs to the glycosphingolipid class and is
EGFR Panitumumab Vectibix overexpressed on the cell surface of neuroblastomas. GD2 was also found to be expressed
HER2 Pertuzumab Perjeta in melanomas, small-cell lung cancer and bone and soft-tissue sarcomas
PDGFRA Olaratumab Lartruvo Platelet-derived growth factor receptor α PDGFRα is a tyrosine kinase receptor expressed
HER2 Trastuzumab Herceptin on hematopoietic cells and cells of mesenchymal origin in central nervous system, gonads,
EGFR Necitumumab Portrazza lung, intestines, skin, and skeleton. It has been aberrantly expressed in several human
cancers and its signaling can contribute to the maintenance of the tumor microenvironment
A main concern with blockage of human epidermal growth factor receptor 2 (HER-2) has been ventricular dysfunction and congestive heart failure. Severe
cardiac side effects were seen in Nezitumumab (cardiopulmonary arrest and/or sudden death in combination with Gemcitabine and Cisplatin).
Hypomagnesemia occurred in 83% and may aggravate cardiac problems. As the drugs are usually used in combination with chemotherapy typical side
effects like anemia, leukopenia, diarrhea, infections, hypothyroidism, metabolic changes, pathological fractures, bone necrosis, convulsions, skin
infections, and kidney failure among others are listed in the drug sheet. The use of mAbs against EGFRs in solid tumors is associated with an increased
risk of FAEs (Li, PlosOne 2013)
Infusion reactions can occur and are as common as 3%. With Cetuximab they can be fatal. Cetuximab is associated with pulmonary and dermatological
toxicity, infections. An acneiform rash appears to be typical of Cetuximab and Panitumumab. Dermatologic toxicities may be complicated by infection
including sepsis as well as abscesses requiring incisions and drainage
Dinutuximab can cause severe pain by irritating nerve cells as well as nerve damage and life-threatening infusion reactions. Common other side-effects
are pyrexia, capillary leak syndrome, hypotension, infections, and sepsis
Olaratumab: Hematological problems like neutropenia in more than 50% of patients and lymphopenia. These will be aggravated with combining the drug
with chemotherapy
T. Niehues
some taken from market, some in development): Surface markers on hematopoietic malignancies
Target Target
system molecule Antibody Brand name Physiology/pathophysiology
CD38 Daratumumab Darzalex CD38: Transmembrane protein, ubiquitously expressed with intra- and
extracellular function, regulates intracellular Calcium. Disease marker for
Lymphomas/Myelomas. Loss of CD38 function is associated with
immunodeficiency, metabolic/behavioral alterations (Malavasi, Physiol. Rev 2008)
CD319 Elotuzumab Empliciti CD319: marker of normal plasma cells
(SLAMF7)
CD19 Blinatumomab Blincyto Present physiologically on immature B-cells in the bone marrow, mature B-cells
and on some plasma cells as well as on many leukemia/lymphoma cells.
Overexpression of CD19 has been linked with development of autoimmunity.
CD30 Brentuximab vedotin (TNF Adcentris CD30: expressed by activated T- and B-cells and belongs to the tumor necrosis
receptor SF8) factor receptor family. It is classically expressed on Hodgkin lymphoma Reed-
Sternberg cells.
CD20 Ibritumomab tiuxetan Zevalin See above, Table 20.13
CD20 Tositumomab Bexxar
CD20 Obinutuzumab Gazyva
CD20 Ofatumumab Arzerra
CD33 Gemtuzumab Ozogamicin Mylotarg CD33 is widely expressed on bone marrow cells, precursor cells of the myeloid
and lineage and expressed in many acute myeloid leukemias (AML)
All monoclonal abs in this group: Will be used in combination before or after chemotherapy so that a clean allocation of drug-specific side-effects is
difficult
Blinatumomab: a cytokine release syndrome can occur and may be life-threatening. Neurological toxicities which may be severe and life-threatening as
well as fatal have been observed (FDA product information)
Blinatumomab: Infusion reactions, interference with blood compatibility testing
Brentuximab: Cases with PML have been reported (Jalan P, Clinical neurologic neurology and neurosurgery 2012;114:1335)
Gemtuzumab/Ozogamicin caused bone marrow suppression with severe neutropenia, severe bacterial and fungal infections. It is unclear whether it is
337
effective against AML and was withdrawn from the market in 2010
338
some taken from market, some in development): Microorganisms, liver cells

Target system Target molecule Antibody Brand name
Microorganisms
F protein of RSV Palivizumab Synagis
Protective antigen of Bacillus anthracis Raxibacumab Raxibacumab
Protective antigen of the Anthrax toxin Obiltoxaximab Anthem
Clostridium difficile toxin B Bezlotoxumab Zinplava
Palivizumab: There have been rare cases of anaphylaxis and hypersensitivity reactions as well as severe thrombocytopenia
Raxibakumab: Only mild to moderate infusion reactions were observed in normal human volunteers similar to Palivizumab
Obiltoxaximab: Hypersensitivity and anaphylaxis have been observed.
Bezlotoxumab: Heart failure was reported more commonly in patients with a history of congestive heart failure. Hypersensitivity, anaphylaxis
Liver cells/LDL-receptors
PCSK9 Evolocumab Repatha
PCSK9 Alirocumab Praluent
There have been no drug specific side effects reported so far in the initial clinical trials. However, both drugs were licensed in 2015 and large-scale
experience is lacking
Drugs
Dabigatran Idarucizumab Praxbind
Hypersensitivity reactions and anaphylaxis may occur as in most of the other monoclonal antibodies
RSV respiratory syncytial virus
PCSK9 Proprotein convertase subtilisin/Kexin type 9 binds to the receptor für LDL (low density lipoprotein) particles
T. Niehues
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Monoclonal Anti-CD20 (B-Cell) Antibody
and Autoimmune Diseases 21
Bertrand Godeau
Abbreviations
AAV ANCA-associated vasculitis

AchR Acetylcholine receptor
AIDs Autoimmune diseases
AIN Autoimmune neutropenia
ANCA Antineutrophil cytoplasmic antibody
APL Antiphospholipid
APS Antiphospholipid syndrome
BAFF B-cell activating factor
CAD Cold-agglutinin disease
CAPS Catastrophic antiphospholipid syndrome
CLL Chronic lymphocyte leukemia
CVID Common variable immunodeficiency syndrome
EGPA Eosinophilic granulomatosis with polyangiitis
GCA Giant cell arteritis
GPA Granulomatosis with polyangiitis
IFNγ Interferon-γ
ITP Immune thrombocytopenia
IVIg Intravenous immunoglobulin
MG Myasthenia gravis
MS Multiple sclerosis
MuSK Muscle-specific tyrosine kinase
PR3 Proteinase 3
PRCA Pure-red cell aplasia
B. Godeau, MD
Internal Medicine Unit, Reference Center of Adults’ Autoimmune Cytopenia,
CHU Henri Mondor, APHP, UPEC, 94010 Créteil, France

344 B. Godeau
pSS Primary Sjögren’s syndrome

RA Rheumatoid arthritis
RBC Red blood cell
SLE Systemic lupus erythematosus
SSc Systemic sclerosis
TNF-α Tumor necrosis factor-α
TPO-Ras Thrombopoietin receptor agonists
wAIHA Warm autoimmune hemolytic anemia
21.1 Introduction
Autoimmune diseases (AIDs) are a heterogeneous group of conditions with diverse

clinical manifestations and complex pathogenesis. Steroids and immunosuppres-
sants are always the cornerstone of treatment of the severe form of most AIDs, but
biologic therapies have become a new weapon in the treatment. Biologic therapies
have deeply changed the natural history of diseases such as rheumatoid arthritis
(RA) and other inflammatory arthritis diseases and antineutrophil cytoplasmic anti-
body (ANCA)-associated vasculitis. However, although life-changing in some
AIDs, the adverse effects accompanying biologic therapy, such as infection and
immunogenicity, and the often disappointing long-term effects with high risk of
relapse remain important issues. We still need to identify new candidate targets.
Context Autoimmune diseases
Immunohematology Immune thrombocytopenia (ITP)
Warm autoimmune hemolytic anemia
(wAIHAI)
Cold-agglutinin disease (CAD)
Autoimmune neutropenia (AIN)
Pure-red cell aplasia (PRCA)
Acquired hemophilia
Acquired thrombocytopenic purpura
(TTP)
Common variable immunodeficiency (CVID) Immune cytopenia (ITP, wAHAI, Evans
syndrome syndrome)
Systemic autoimmune diseases and connective Systemic lupus erythematosus (SLE)
tissue diseases Antiphospholipid syndrome (APS)
Sjögren’s syndrome
Systemic sclerosis (SSc)
Inflammatory muscles diseases
Rheumatoid arthritis
Systemic vasculitis ANCA-associated vasculitis
Cryoglobulinemia
Giant cell arteritis and Takayasu arteritis
Nervous system Multiple sclerosis (MS)
Myasthenia gravis
21 Monoclonal Anti-CD20 (B-Cell) Antibody and Autoimmune Diseases 345
B cells play an important role in the immune response (Godeau and Stasi 2014).
Their major role is to produce antibodies. B cells are also efficient antigen-presenting
cells for T cells and secrete various cytokines such as interleukin-1 (IL-1), IL-10,
IL-6, interferon-γ (IFNγ), and tumor necrosis factor-α (TNF-α), which activate
macrophages, dendritic cells, and immunoregulatory cells. B cells have an impor-
tant role in the pathophysiology of AIDs, and a rational approach to AID treatment
involves B-cell depletion.
Human CD20 is a non-glycosylated phosphoprotein exclusively expressed in the
B-cell hematopoietic lineage but not in hematopoietic stem cells or plasma cells.
Rituximab is a human-to-mouse chimeric anti-CD20 monoclonal antibody that
induces rapid, profound, and prolonged B-cell depletion. It was initially developed
20 years ago to treat lymphoma but is now used to treat various AIDs.
Here, we discuss the use of rituximab in treatment of the most frequent AIDs.
21.2 Rituximab and AIDs
21.2.1 Immunohematology and Hemostasis
21.2.1.1 Immune Thrombocytopenia (ITP)
Clinical Manifestations
ITP is a heterogeneous disease with a complex pathophysiology, involving enhanced
platelet clearance as well as impaired platelet production. ITP remains a diagnosis
of exclusion, and primary (or isolated) ITP and secondary ITP are commonly dif-
ferentiated. ITP manifests by bleeding, which usually occurs with platelet count
<30 G/L.
Rules of Treatment
Steroids and intravenous immunoglobulin (IVIg) are the first-line treatments. They
are highly effective, but relapse occurs in most adults, and second-line treatments
are frequently required. Second-line treatments include thrombopoietin receptor
agonists (TPO-RAs), dapsone, danazol, rituximab, and splenectomy. Each approach
has benefits and risks that should be considered carefully. Unlike the availability of
international guidelines, consensus remains lacking on the treatment of ITP and
should be personalized.
Results of Rituximab
More than 15 years ago, in an open prospective study, Stasi et al. (2001) gave ritux-
imab to adults with chronic ITP according to the infusion regimen used for lym-
phoma (i.e., 4 weekly infusions of 375 mg/m2 rituximab). They reported an overall
response rate of 52%. In view of these encouraging results, other groups conducted
uncontrolled studies. In a prospective multicenter registry of 248 adult patients with
ITP treated with rituximab, 61% showed an overall initial response and at a median
follow-up of 24 months, and 39% showed a lasting response (Khellaf et al. 2014).
346 B. Godeau
The pattern of response was similar with the two rituximab regimens (four infusions
of 375 mg/m2 and two fixed 1-g infusions 2 weeks apart), and reassuring data were
obtained on the safety of the treatment. In a prospective double-blind randomized
study, Ghanima et al. (2015) compared rituximab with placebo and standard care as
a second-line treatment for ITP without splenectomy. The rate of complete response
at 24 weeks was greater with rituximab than placebo, with a trend toward a lower
rate of splenectomy in the rituximab arm. However, rituximab did not reduce the
rate of treatment failure. The modest long-term effect of rituximab was confirmed
in a retrospective study of 72 adults and 65 children finding 5-year estimates of 21%
and 26% persistent response, respectively (Patel et al. 2012).
Comments and Perspectives
With its benefit/risk ratio, rituximab used off-label is a valid option for treating per-
sistent or chronic ITP. However, relapses are frequent, and the long-term response
appears modest. Therefore, strategies to ameliorate the long-term efficacy of the
treatment must be developed. Several options that may be tested include giving
rituximab first or early on after ITP diagnosis, maintenance treatment with repeated
infusions, and combining rituximab with other treatments such as dexamethasone
or anti-B-cell activating factor (BAFF, also called BLyS) monoclonal antibody for a
synergistic effect.
21.2.1.2 Warm Autoimmune Hemolytic Anemia (wAIHA)
AIHA is a rare AID in which autoantibodies directed toward red blood cell (RBC)
antigens lead to their accelerated destruction. The diagnosis of AIHA mainly relies
on the direct antiglobulin test. The classification of AIHA is based on immuno-
chemical properties and especially on the thermal amplitude of the autoantibody
(“warm” or “cold” type), which in clinical practice mainly relies on the interpreta-
tion of the direct antiglobulin test pattern but also on the presence or not of an
underlying condition or disease (i.e., secondary versus primary AIHA). The distinc-
tion between AIHA due to warm antibody (wAIHA) and cold antibody is crucial
because it affects both the prognosis and treatment.
wAIHA can be isolated or associated with various diseases, including mainly
systemic lupus erythematosus (SLE) and chronic lymphocyte leukemia (CLL).
The clinical presentation of AIHA is usually subacute and may be rather insidi-
ous anemia. An abrupt onset with the presence of dark reddish urine reflecting the
presence of intravascular hemolysis and hemoglobinuria is rarely observed.
Rules of Treatment
RBC transfusion is indicated in patients with disabling symptoms of anemia and/or
a serious underlying cardiovascular condition.
The first-line treatment of primary wAHAI remains corticosteroids given at an
initial daily dose of 1–1.5 mg/kg. The total duration of treatment lacks consensus,
but the likelihood of early relapse is very high if the treatment is prematurely
stopped, so corticosteroids should be maintained at least 3 months after a complete

response.
Management for stage A CLL and active hemolysis should be as for primary
wAIHA. However, progressive CLL must be treated more aggressively, with com-
bined regimens such as rituximab + cyclophosphamide and dexamethasone
(R-CDex).
Two open randomized controlled studies (Barcellini et al. 2012; Birgens et al. 2013)
and one double-blind controlled study (Michel et al. 2017) using a different pattern
of rituximab administration gave promising data and demonstrated the efficacy of
rituximab in primary wAHAI. In a prospective study with a placebo, the overall
response rate at 1 year was 75% with rituximab versus 31% with placebo.
The data from the literature support, whenever possible, the use of rituximab off-
label for patients refractory to corticosteroids and those with a chronic active and/or
relapsing primary wAIHA who need to be maintained on prednisone (or predniso-
lone) at a daily equivalent or >15 mg to maintain at least partial remission.
21.2.1.3 Cold Agglutinin Diseases (CADs)
Primary chronic CAD is a clonal lymphoproliferative B-cell bone-marrow disorder.
The immune hemolysis is complement dependent, mediated by activation of the
classical pathway and phagocytosis of erythrocytes opsonized with complement
protein C3b. CAD can be an indolent disease, but typical clinical features include
episodes of transient anemia that can be acute and severe and frequently associated
with cold-induced ischemic symptoms ranging from mild to disabling.
Rules of Treatment
In the indolent disease form, no treatment is required, but patients should avoid
exposure to cold. Pharmacologic treatment should be offered for s ymptom-producing
anemia or disabling circulatory symptoms. Corticosteroids usually give disappoint-
ing results and the indication of steroids for CAD is still debatted. Splenectomy is
not a good option because hemolysis in CAD is intravascular. Successful CAD
therapy targets the pathogenic B-cell clone.
Rituximab monotherapy can induce partial remission in about 50% of patients
(Berentsen 2013; Berentsen et al. 2017). Those with relapse after previous treatment
with rituximab may respond to a second or even a third series of monotherapy, and
the treatment is well tolerated. Despite a somewhat disappointing median response
duration of about 1 year, single-agent therapy with rituximab should still be consid-
ered first-line treatment in some patients.
348 B. Godeau
Combination therapy with fludarabine and rituximab is more efficient, resulting in
remission in approximately 75% of patients and complete response in 20% and a
median response duration of more than 5 years. However, because of the toxicity
profile, this treatment should be reserved for the more severe forms of CAD in
patients showing failure of remission with rituximab monotherapy. Recent study
suggests that Bendamustine could also be a good option.
In the near future, complement-modulating agents seem promising.
21.2.1.4 Autoimmune Neutropenia (AIN)
AIN is a rare and heterogeneous group of diseases with variable clinical manifes-
tations from asymptomatic to severe forms associated with infectious complica-
tions (Autrel-Moignet and Lamy 2014). It is caused by antibodies directed against
neutrophil-specific antigens. It includes primary and secondary autoimmune neu-
tropenia. Acute autoimmune neutropenia can be related to drug-induced mecha-
nisms or viral infections. Chronic autoimmune neutropenias occur in the context
of AIDs such as SLE or Sjögren’s syndrome, hematologic malignancies such as
large granular lymphocyte leukemia, primary immune deficiency syndromes, or
solid tumors.
Rules of Treatment
The therapeutic management depends on the etiology. Granulocyte growth factor is
an option and can be transiently used in cases of symptomatic profound neutrope-
nia. The question of their long-term safety is debated. Corticosteroids or immuno-
suppressive therapy (mainly cyclophosphamide, methotrexate, or cyclosporine) are
indicated in infection-related autoimmune neutropenia or with symptomatic auto-
immune disease or large granular lymphocytic leukemia.
Rituximab has been only occasionally used, with disappointing results, and does
not seem a valid option except when autoimmune neutropenia is associated in a
setting of Evans syndrome (association of autoimmune neutropenia with ITP and/
or wAHAI).
Of note, transient profound neutropenia, which is usually asymptomatic, is a
complication of rituximab. This adverse event is mainly observed when rituximab
is used for treating CLL or malignant lymphoma but is rarely observed in the set-
ting of AIDs.
21.2.1.5 Pure-Red Cell Aplasia (PRCA)
PRCA is a rare syndrome caused by isolated erythropoietic hypoplasia with severe
normocytic and reticulocytopenic anemia and a normally cellular bone marrow but
devoid of erythroblasts. PRCA is often diagnosed in conjunction with a variety of

diseases, such as lymphoproliferative disorders (mainly CLL), viral infections (par-
vovirus B19, HIV), wAHAI, rheumatologic disorders, and allogeneic stem cell
transplantation.
Rules of Treatment
Acquired PRCA is managed as an AID, by immunosuppressive therapy with corti-
costeroids and cyclosporine A as first-choice treatments.
Only isolated case reports suggested that rituximab could be effective in PRCA. Good
response has been reported in a setting of PRCA associated with various diseases
such as SLE or CLL or after allogenic bone-marrow transplantation.
Rituximab should be reserved as rescue treatment for patients who are refractory to
steroids and immunosuppressive therapy such as cyclosporine A, and it cannot be
considered as first-line treatment (Tendas et al. 2016).
21.2.1.6 Acquired Hemophilia
Acquired hemophilia is an AID caused by the development of specific autoanti-
bodies that inhibit factor VIII (FVIII). It is associated with a high mortality rate,
usually between 10 and 30%. Hemorrhagic manifestations of acquired hemophilia
occur as acute, spontaneous, or traumatic in patients with no prior history of
bleeding. The most common clinical findings are profuse cutaneous bleeding.
Acquired hemophilia can complicate pregnancy and can be associated with many
diseases such as various AIDs (SLE, Sjögren’s syndrome, etc.), hematological
diseases including lymphoid hemopathy, myelodysplastic syndrome, solid tumors,
chronic viral infection (hepatitis B and C virus [HBV, HCV]), and allergic reac-
tion to drugs.
Rules of Treatment
It consists of two parts: treatment targeting abortion or preventing bleeding epi-
sodes and that aimed at eradicating the autoantibody. As bypassing agents, two
drugs are used for this indication: recombinant activated factor VII (rFVIIa
[NovoSeven®]) and activated prothrombin complex concentrates (APCC
[FEIBA®]).
The first-line immunosuppressive treatment most commonly used and recom-
mended is based on corticosteroids (1 mg/kg/day) alone or associated with cyclo-
phosphamide given at low doses (1–2 mg/kg/day), for between 3 and 5 weeks. The
combination of immunosuppression and comorbidities, due to patient age and
comorbidities, leads to the occurrence of cytopenias and secondary infections in
almost half of all patients.
350 B. Godeau
Most reviews and guidelines point to rituximab as the alternative of choice with fail-
ure of first-line treatment. The usual pattern involves the administration of weekly
doses of 375 mg/m2/week for 4 weeks. Rituximab requires several weeks or months
to achieve eradication of the inhibitor. For the more severe forms of acquired hemo-
philia, particularly with high titers of inhibitors, some authors consider that rituximab
should be combined with other immunosuppressive drugs (Collins et al. 2012).
A large controlled prospective study is in progress in France to better define the
place of rituximab in primary acquired hemophilia.
21.2.1.7 Acquired Thrombotic Thrombocytopenic Purpura (TTP)
Acquired autoimmune TTP is a severe form of thrombotic microangiopathy charac-
terized by the association of a microangiopathic hemolytic anemia with a peripheral
thrombocytopenia, organ failure of variable severity due to thrombi in microvascu-
lature, and antibody-mediated severe deficiency (<10% of normal activity) in the
von Willebrand-factor-cleaving protease ADAMTS13.
TTP can complicate pregnancy and can be associated with many diseases such as
various AIDs (SLE, Sjögren’s syndrome, etc.), viral infection (HIV, etc.), reaction
to drugs (cyclosporine A), and bone-marrow transplantation.
Rules of Treatment
Daily therapeutic plasma exchange, which addresses the ADAMTS13 deficiency
and to a lesser extent removes serum anti-ADAMTS13 antibodies and possibly pro-
aggregant substances, has transformed the prognosis of TTP, with current overall
survival rates of 80–85%. Because acquired TTP is now considered an AID, immu-
nosuppressive drugs are also involved in the therapeutic strategy. In addition to the
development of plasma exchange, rituximab has been a major breakthrough in man-
aging this disease.
For the acute phase of TTP, most studies reported that remission was achieved in most
cases, typically in <4 weeks (Froissart et al. 2015). Rituximab is now routinely recom-
mended during the acute phase, typically in patients with a suboptimal response to
plasma exchange, or even as a first-line therapy. However, whether rituximab should
be reserved for patients with suboptimal response to standard treatment or used as a
first-line therapy for all patients with autoimmune TTP is still debated.
At least 40% of patients experience a recurrence of TTP. Each relapse exposes the
patient to risk of death and to complications related to plasma exchange. ADAMTS13
activity represents a reliable marker of disease activity because patients who remain
with severe enzyme deficiency are at high risk of relapse. Preliminary reports sug-
gested that infusions of rituximab could prevent TTP relapse in patients with severe
persistent ADAMTS13 deficiency and otherwise in clinical and hematologic remis-
sion. These findings argue for regular systematic assessments of ADAMTS13 activ-
ity during follow-up, to identify at an early stage those patients at risk of relapse.
Preemptive rituximab represents a promising strategy that could modify the long-
term prognosis.
21.2.2 Autoimmune Manifestations of Common Variable

Immunodeficiency (CVID) Syndrome
21.2.2.1 Clinical Manifestations

CVID is a primary immune deficiency characterized by reduced serum levels of
IgG, IgA, and/or IgM with reduced or absent specific antibody production. The
diagnosis is typically between the ages of 20 and 40 years, but about 20% of patients
are younger than 20. Infectious complications are the disease hallmark, but in two
thirds of patients, granulomatous disease or one or more inflammatory and autoim-
mune manifestations develop. Inflammatory manifestations involve mainly the lung
with progressive chronic lung diseases. ITP and wAHAI are the most common
AIDs and complicate the CVID outcome. Lymphoma or other malignancies can
occur in about 10% of patients.
21.2.2.2 Rules of Treatment

Substitutive treatment based on repeated IVIg infusion is the cornerstone of CVID
and prevents infectious complications. However, this treatment is ineffective in
treating autoimmune manifestations and particularly ITP and wAHAI. In this case,
prolonged treatment with steroids and splenectomy should be avoided because of
the risk of infection.
21.2.2.3 Results of Rituximab

In a retrospective study of 33 patients with CVID and ITP and/or wAHAI treated
with rituximab, the overall initial response rate to rituximab was 85% (Gobert et al.
2011). After a mean follow-up of more than 3 years, ten of the initial responders
showed relapse, and retreatment with rituximab was successful in seven out of nine
responders. Severe infections occurred after rituximab in eight adults (24%), four
not on IVIg replacement therapy. In conclusion, rituximab appears to be highly
effective and relatively safe for managing CVID-associated severe immune cytope-
nias. However, it should be systematically associated with IVIg replacement to limit
the risk of infectious complications.
21.2.2.4 Comments and Perspectives

TPO-RAs are very effective in primary ITP. They could be a good option for treat-
ing ITP associated with CVID. The respective places of TPO-RAs and rituximab in
this setting are still debated.
352 B. Godeau
21.2.3 Systemic Autoimmune Diseases and Connective Tissue

Diseases: “Lupus Group”
21.2.3.1 Systemic Lupus Erythematosus (SLE)
SLE is a chronic autoimmune condition with unpredictable course, intermingled
with flares and periods of remission. It can affect the skin, muscles and articles,
heart, lung, kidney, and peripheral and central nervous system. Blood manifesta-
tions are frequent, and autoimmune cytopenias, including ITP and wAIHA, can
be severe.
Rules of Treatment
Treatments may include nonsteroidal anti-inflammatory drugs, corticosteroids,
immunosuppressants, and hydroxychloroquine. Although the prognosis has
improved in the past decades, current therapies are still associated with treatment-
related complications. Recently, there has been major progress in understanding the
pathogenesis of SLE, paving the way for the development of new biologic agents,
potentially revolutionizing the treatment of SLE.
The use of rituximab in patients with SLE has been investigated in two random-
ized controlled trials, EXPLORER (the Exploratory Phase II/III SLE Evaluation
of Rituximab) (Merrill et al. 2010) and LUNAR (Lupus Nephritis Assessment
with Rituximab) (Rovin et al. 2012), with negative results regarding superiority to
conventional treatment. However, before concluding that rituximab is not effec-
tive in SLE, a critical evaluation of the design of the EXPLORER and LUNAR
trials is required. The results of these two trials suggested that the use of rituximab
in SLE may be controversial, but it is still extensively used “off-label,” especially
in cases refractory to standard treatment (Cobo-Ibanez et al. 2014; Duxbury et al.
2013; Sciascia et al. 2015). Rituximab is effective in autoimmune cytopenia asso-
ciated with SLE, with severe kidney involvement in case of failure of “conven-
tional treatment” such as cyclophosphamide, mycophenolate mofetil, and
azathioprine. A prospective, observational, single-center cohort study evaluated
the effectiveness of treating lupus nephritis with rituximab and mycophenolate
mofetil but no oral steroids (Condon et al. 2013). After 1 year of follow-up, over-
all response rate was >80%, which demonstrates that oral steroids can be safely
avoided in treating lupus nephritis.
Rituximab could be associated with risk of fatal multifocal progressive leukoen-
cephalopathy due to JC virus infection in the setting of SLE treated with rituximab.
However, this risk appears exceptional.
Today, unlike in the absence of a license, rituximab can be proposed for SLE
patients with lupus nephritis resistant to conventional treatment or autoimmune
cytopenia.
Other B-cell-targeted therapies that inhibit BAFF currently being assessed include
belimumab, tabalumab, and blisibimod. Belimumab received a license for non-
severe SLE (without central system or kidney involvements).
21.2.3.2 Antiphospholipid Syndrome (APS)
APS is characterized by recurrent thrombosis and/or obstetric complications with
the presence of antiphospholipid antibodies (lupus anticoagulant, anti-cardiolipid
antibodies, anti-beta2-GP1 antibodies). It can be isolated and considered “primary”
or associated with SLE.
The most frequent clinical manifestation is deep venous thrombosis, whereas
cerebrovascular accident is the most prevalent manifestation of arterial thrombosis.
Fetal losses (early and late), prematurity, and preeclampsia are the most frequent
obstetric manifestations. The catastrophic variant of the antiphospholipid (APL)
syndrome (CAPS) is characterized by thrombosis in multiple organs developing
over a short time. The prognosis of CAPS is severe, and mortality remains high.
Rules of Treatment
The treatment for thrombotic APS is based on control of vascular risk factors, ace-
tylsalicylic acid as primary thromboprophylaxis, and long-term anticoagulant treat-
ment as secondary thromboprophylaxis.
The combined treatment with anticoagulation therapy plus glucocorticoids plus
plasma exchange and/or IVIg results in a high recovery rate in patients with CAPS.
For APS, we have only few data focused on the interest of rituximab in this setting.
Some case reports suggested that rituximab could be effective for treating some
non-thrombotic manifestations associated with APS, such as thrombocytopenia of
skin necrosis (Ponsa et al. 2015).
For CAPS, despite the reduced mortality with treatment, some patients are
refractory, such as those who die despite first-line treatments or those with recurrent
episodes of CAPS. A review of the literature reported 20 patients with CAPS treated
with rituximab (Berman et al. 2013). The number of patients was too low to draw
firm conclusions, but 75% of patients recovered from the acute CAPS episode and
20% died. These results suggest that rituximab could have a role in treating APL-
positive patients, especially those with refractory CAPS.
21.2.3.3 Sjögren’s Syndrome
Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease that involves
the exocrine glands and internal organs. pSS leads to the destruction and loss of
354 B. Godeau
secretory function due to intense lymphoplasmacytic infiltration. In most patients,

the outcome is benign, and symptoms are limited to sicca syndrome. However, in
some patients, pSS may have systemic involvement, including the pulmonary, renal,
vascular, central, and/or peripheral nervous systems. Autoimmune cytopenia is a
known complication. The risk of non-Hodgkin lymphoma is higher than in the gen-
eral population.
Rules of Treatment
Therapeutic options include mainly symptomatic and supportive measures, and tra-
ditional immunosuppressant drugs have shown no effectiveness in randomized tri-
als. The use of systemic therapies for dryness, chronic pain, or fatigue is not
warranted. The management of pSS should be organ specific, with low-dose ste-
roids in patients with moderate systemic activity, limiting the use of high-dose ste-
roids and second-line therapies to refractory or potentially severe cases.
The number of published articles on rituximab used to treat pSS has been grow-
ing. However, most identified studies are case reports or series of specific sys-
temic manifestations, and only a few randomized studies of rituximab have
compared the effectiveness of this drug to placebo or other drugs (Souza et al.
2016). Because of multiple biases due to the design of most of these studies,
drawing firm conclusions is difficult. The effect on improving lacrimal gland
function appears modest, and we have no proof of the potential of this drug for
improving salivary flow. Also no level of evidence suggests improvement of oral
dryness. In particular, a double-blind prospective controlled study conducted in
France that compared rituximab and placebo and included 120 patients confirmed
that rituximab did not alleviate symptoms or disease activity in patients with pSS
at week 24, although it alleviated some symptoms at earlier time points
(Devauchelle-Pensec et al. 2014).
Rituximab is not indicated in pSS but can be discussed for some extraglandular
manifestations such as autoimmune cytopenia.
21.2.3.4 Systemic Sclerosis (SSc)
SSc is characterized by diffuse microangiopathy and accumulation of collagen and
other matrix constituents in the skin and target internal organs. Typical SSc symp-
toms can be skin ulcers, pulmonary arterial hypertension, and/or renal scleroderma
crisis with fibrotic cutaneous and visceral organ involvement affecting particularly
the heart and lung. Autoimmunity may contribute to both vascular and fibrotic SSc
manifestations.
Rules of Treatment
The effect of nonselective immunosuppressive treatments, usually used during the
early phases of SSc to control skin and lung inflammation, is often unpredictable.
High-dose steroids should be avoided because of the risk of severe renal sclero-
derma crisis. These treatments tend to lose their efficacy once the disease enters a
chronic phase, and consequently long-term treatment is not recommendable, con-
sidering the potential severe side effects.
Small open studies and case reports suggested that rituximab could act on skin and
lung fibrosis (Giuggioli et al. 2015). However, multiple biases are present, so one
cannot conclude on the interest of rituximab in SSc. A multicenter prospective case-
control study conducted in Europe included 63 patients receiving rituximab (Jordan
et al. 2015). The comparison of rituximab-treated versus untreated matched-control
SSc patients demonstrated improved skin fibrosis and prevention of worsening lung
fibrosis, which supports the therapeutic concept of B-cell inhibition in SSc.
The results of the European Scleroderma Trial and Research (EUSTAR) group
remain preliminary and need to be viewed with caution, recognizing the spontane-
ous regression of skin thickening that may occur early during the disease, and
other studies are required before proposing rituximab as first-line treatment for
severe pSS.
21.2.3.5 Inflammatory Muscle Diseases
To date, four main groups of idiopathic inflammatory myopathies (IIMs) have been
identified—polymyositis, dermatomyositis, immune-mediated necrotizing myopa-
thy, and sporadic inclusion body myositis—on the basis of clinical presentation and
muscle pathology. Important phenotypical differences (muscular and/or extramus-
cular manifestations) persist within a group. We now have routine access to assays
for detecting different antibodies, and all groups of myositis may present one of
those autoantibodies. Most allow for identifying homogenous patient groups more
precisely than with the classical international classifications of myositis.
Rules of Treatment
High-dose steroids remain the first-line treatment for inflammatory muscle diseases
except for sporadic diseases including body myositis, for which steroids are ineffec-
tive. IVIg is indicated, particularly in dermatomyositis associated with dysphagia.
Immunosuppressive treatment such as methotrexate, mycophenolate mofetil, and
cyclophosphamide could be associated with steroids as a first-line treatment in the
more severe forms of diseases or in patients refractory to steroids.
356 B. Godeau
We have only few data on the effectiveness of rituximab in IIMs. Most of the
reported studies are retrospective and included a small number of patients. Moreover,
they grouped different types of IIMs. Drawing firm conclusions is difficult, and the
exact role of rituximab in the therapeutic strategy of IIMS is still a matter of debate,
but these data suggest that rituximab could be effective in all IIMs (Hervier and
Benveniste 2015). The delay of action is sometimes long, and at least 4 months may
be needed before a response is seen. Rituximab should not be proposed alone but
should be associated with steroids and/or immunosuppressive drugs. Relapses are
frequent. In case of relapse, a new course of rituximab could be proposed.
Despite these relatively modest results, rituximab may play a role in the treatment
of IIMs. All other available biotherapies such as anti-IL-1, anti-IL-6, and anti-IFN-α
therapies have been not studied, and anti-TNF-α agents are contraindicated with
myositis, at least in patients with positive myositis-specific autoantibodies.
21.2.3.6 Rheumatoid Arthritis (RA)
RA is a systemic autoimmune disease characterized by joint inflammation that often
evolves into erosive joint damage with significant disability.
Rules of Treatment
Methotrexate remains the first-line treatment. Prolonged steroid treatment should be
avoided. The development of anti-TNF agents has completely revolutionized the
natural history of the disease and should be rapidly associated with methotrexate in
cases of lack of response.
Some patients show an inadequate response to anti-TNF agents. Rituximab is indi-
cated in such cases (Cohen and Keystone 2015; Rossi et al. 2015. Several random-
ized controlled prospective studies compared two infusions of rituximab 1000 mg
to placebo. Methotrexate was used in both. Treatment with rituximab has been
clearly demonstrated as more effective than placebo in treatment-naïve patients and
those with anti-TNF treatment failure. Rituximab is more effective in “seropositive”
patients. It has been studied in combination with anti-TNF agents, and the numeri-
cal risk of serious adverse events was only slightly increased but without a signifi-
cant increase in efficacy. The optimal rituximab dose is controversial. The
2 × 1000- and 2 × 500-mg doses may be equivalent in terms of improvement in
signs and symptoms, but the 2 × 1000-mg dose showed better outcomes and should
be used. Relapses are frequent, and the duration of the effect is quite variable. So,
the optimal timing for retreatment is difficult to predict. A review of retreated
patients from the clinical trials suggested that the fixed-interval (24 week) treat-to-
target strategy was superior to retreatment at the discretion of the physician. The
latest European consensus statement suggests that retreatment in initial responders

should be considered at 24 weeks for patients who do not achieve low disease activ-
ity or remission and that it should be delayed otherwise until disease activity flares.
The incidence of human anti-chimeric antibodies varied from 2.7 to 7.1%, but
the clinical significance of the antibodies is still debated.
Rituximab has been a significant addition to the short list of biologic agents approved
for treating RA. The safety is reassuring, but as with all biologics for RA, further
information regarding the safety of rituximab over longer periods is critical.
21.2.4 Systemic Vasculitis
21.2.4.1 ANCA-Associated Vasculitis (AAV)
AAV includes granulomatosis with polyangiitis (GPA), eosinophilic granulomato-
sis with polyangiitis (EGPA), and microscopic polyangiitis (MPA), which are small-
vessel vasculitides associated with the presence of ANCAs to proteinase 3
(PR3-ANCAs) or myeloperoxidase (MPO-ANCAs). The clinical manifestations of
GPA range from limited upper respiratory tract inflammatory disease to severe
lower respiratory tract, renal, and nervous system vasculitis. MPA involves necro-
tizing systemic vasculitis of the respiratory tract, kidneys, and nervous system.
EGPA clinical manifestations include asthma, nasal polyps, peripheral blood eosin-
ophilia, and systemic vasculitis of the respiratory tract, skin, heart, and nervous
system.
Rules of Treatment
The management of AAV is in accordance with the disease severity and is based on
extensive clinical trial and clinical practice data. High-dose steroids associated with
immunosuppressive drugs (mainly cyclophosphamide as attack treatment and meth-
otrexate or azathioprine as maintenance treatment) have been considered the corner-
stone for treating the most severe forms for a long time. Plasma exchange is also
indicated in life-threatening situations such as pulmonary hemorrhage or acute renal
failure.
In two multicenter, prospective, randomized controlled studies, at 6 months, ritux-
imab was found to be not inferior to cyclophosphamide for inducing remission of
GPA and MPA. From these two studies, the US Food and Drug Administration
accorded marketing authorization for rituximab as remission-induction and mainte-
nance treatment for these two AAVs. In Europe, rituximab has been authorized for
only induction therapy. Now, rituximab is considered as a first-line treatment, even
for life-threatening GPA and MPA (Lutalo and D’Cruz 2015), except in France,
358 B. Godeau
where the recommendation is to continue to use cyclophosphamide for severe acute

renal failure or pulmonary hemorrhage (Charles et al. 2013). For maintenance treat-
ment, rituximab has been found superior to azathioprine. A fixed-interval rituximab
protocol, with a single 1-g infusion administered every 6 months for 2 years, has
been shown to reduce the rate of clinical relapse as compared with rituximab retreat-
ment at the time of relapse in patients with severe relapsing, refractory AAV.
Indications
For EGPA, studies comparing conventional treatment and rituximab are in progress.
To date, rituximab is not recommended in this setting, but some case reports and
retrospective data suggest that rituximab could be effective in severe cases refrac-
tory to conventional treatment, with systemic vasculitis as the predominant clinical
manifestation.
Rituximab has clearly completely revolutionized the therapeutic strategy of GPA
and MPA. However, relapses are not rare, and the best maintenance treatment with
repeated infusions or combining rituximab with other treatments should be
determined.
21.2.4.2 Cryoglobulinemia
Cryoglobulinemia is characterized by the presence of cryoglobulins in serum. It has
two main subgroups: type I, in which the cryoglobulins are monoclonal, and types
II and III, in which the cryoglobulins are composed of a mixture of monoclonal IgM
and polyclonal IgG (type II) or only polyclonal IgG (type III). Type I is seen exclu-
sively in clonal hematologic diseases, whereas type II/III, named “mixed” cryo-
globulinemia, is seen in hepatitis C virus infection and systemic diseases such as
B-cell hemopathy and connective tissue disorders.
Clinical manifestations are various and include arthralgia, skin purpura, skin
ulcers, renal involvement, and peripheral neuropathy. Life-threatening manifesta-
tions with heart or central nervous system involvement are rarely seen. Occasionally,
when the cryocrit is high, hyperviscosity syndrome may occur, with oronasal bleed-
ing, blurred vision, deafness, headache, confusion, and heart failure.
Rules of Treatment and Place of Rituximab

Only symptomatic cryoglobulinemia should be treated (Muchtar et al. 2017).
For the rare severe forms with hyperviscosity syndrome, plasma exchange is
indicated.
Treating the underlying cause is important. Thus, in type I, the major goal is to
treat the clonal hematologic disease, and in this field, chemotherapy is the corner-
stone of treatment. In this case, rituximab is rarely indicated and may cause IgM
flare and development of hyperviscosity syndrome.
For mixed cryoglobulinemia, with HCV infection, antiviral treatment is indi-

cated. Rituximab can be associated with antiviral therapy in the most severe forms.
In noninfectious mixed cryoglobulinemia, with severe manifestations, treatment
is required (i.e., cutaneous ulcers, glomerulopathy, debilitating neuropathy). The
respective indications of rituximab associated with steroids or cyclophosphamide
plus steroids are debated.
21.2.4.3 Giant Cell Arteritis (GCA) and Takayasu Syndrome
GCA is a large-vessel vasculitis characterized by a granulomatous involvement of
the aorta and/or its major branches that usually affects people older than 50 years. It
is frequently associated with polymyalgia rheumatica and manifests by headache
and proximal myalgia. There is a risk of arteritic ischemic optic neuropathy, which
can result in blindness. In one quarter of patients, the aorta and its major branches
are involved.
Takayasu arteritis is a rare large-vessel vasculitis affecting the aortas and its large
branches including proximal portions of renal, coronary, and pulmonary arteritis. It
affects young patients (younger than 40 years), and it manifests by claudication of
the extremities, myalgia, decreased brachial artery pulse over the subclavian, or
abdominal aorta. Stroke is a complication of the most severe forms.
Rules of Treatment
Steroids remain the first-line treatment of GCA and Takayasu arteritis.
Immunosuppressive drugs are indicated with lack of response to steroids or with
relapse when decreasing the steroids dose. Methotrexate is the immunosuppressive
treatment most frequently used in this setting. Recently, biologic therapy based on
tocilizumab, an anti-IL-6 monoclonal antibody, showed promising results.
We have only a few data for a small number of patients with Takayasu arteritis
treated with rituximab (Loricera et al. 2015). In view of the small number of cases,
we cannot draw definite conclusions, and so far the role of rituximab in the thera-
peutic strategy of ANCA-negative systemic vasculitis is marginal.
21.2.5 Nervous System
21.2.5.1 Multiple Sclerosis (MS)
MS is a chronic inflammatory demyelinating disease caused by an autoimmune
response against central nervous system structures. MS attacks the myelin of the
brain and spinal cord, causing inflammation and often damaging the myelin in
360 B. Godeau
patches. The disease results in a wide variety of symptoms such as dizziness, blad-
der dysfunction, gait, optic neuritis, and sensory impairment, depending on what
part or parts of the central nervous system are affected. The symptoms improve
during periods of remission.
Rules of Treatment
Treatment is very complex and should be personalized. Many treatments now avail-
able include IFN-β, teriflunomide, fingolimod, mitoxantrone, and monoclonal anti-
bodies such as natalizumab and alemtuzumab. The choice of drug used for initial
therapy or escalation of therapy should be based on a benefit/risk evaluation and
tailored to the individual patient’s requirements. Patients should ideally receive
treatment by a specialized multidisciplinary team.
Results of Anti-CD20 Antibody Therapy

There is evidence for B-cell involvement in the pathophysiology of MS, and at the
present, three therapeutic monoclonal antibodies are in clinical phase II and III
trials (rituximab, ocrelizumab, and ofatumumab) (Bittner et al. 2017). Recent
controlled studies demonstrated that ocrelizumab which is a monoclonal antibody
directed against B cells was more effective than IFN (Hauser et al. 2017;
Montalban et al. 2017).
21.2.5.2 Myasthenia Gravis (MG)
MG is an autoimmune neuromuscular junction disorder. It manifests by fluctuating
fatigable weakness involving specific muscle groups. Ocular weakness with asym-
metric ptosis and diplopia is the most common presentation. Oropharyngeal and
limb weakness are less common. Antibodies against acetylcholine receptor (AchR)
or muscle-specific tyrosine kinase (MuSK) are always present and are useful for the
diagnosis.
Rules of Treatment
Treatment must be individualized. Cholinesterase inhibitors are the first-line treat-
ment. Immunosuppressive treatment including steroids, azathioprine, cyclosporine,
or mycophenolate mofetil is indicated in the most severe disabling forms.
Thymectomy can be occasionally indicated. Plasma exchange or IVIg is required in
an emergency for myasthenic crisis and life-threatening situations.
Tandan et al. (2017) recently reviewed the efficacy and safety of rituximab in 169
MG patients from case reports and series. A response was observed in 72% of
patients with MuSK antibodies and in only 30% with AchR antibodies. More than
50% of patients could have a relapse within a mean of 1.5 years after rituximab infu-
sions (Robeson et al. 2017). These results suggest that rituximab could be an attrac-
tive option in severe MG.
Unlike these promising results, experts who recently published international con-
sensus guidance for MG management concluded that no formal consensus could be
reached concerning the place of rituximab in the therapeutic strategy.
Conclusions
Rituximab is used frequently for treating most AIDs. It has deeply changed the
treatment strategy in AAV, and a license was obtained for the treatment of
RA. However, for most AIDs, the drug is used off-label, and randomized con-
trolled studies are often lacking. It is well tolerated, even if infectious complica-
tions are possible, particularly if associated with steroids. One important caveat
is the risk of relapse. For the future, how to obtain better long-term results
remains a crucial issue. Maintenance treatment with repeated rituximab infu-
sions, association of rituximab with other biologic therapies or dexamethasone,
and recognition of predictors of long-term sustained response could be different
options to better select patients with AIDs who could receive rituximab and to
hope for better long-term results.
References
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Paul Imbach - Antibody Therapy-Springer International Publishing (2018)

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ISBN 978-3-319-68037-8 ISBN 978-3-319-68038-5 (eBook)

Library of Congress Control Number: 2017954301

© Springer International Publishing AG, part of Springer Nature 2018

Printed on acid-free paper

 Adrian Newland, CBE, MA, FRCP, FRCPath

1 The Clinical Translation of Intravenous Immunoglobulin

Part I Update of Substitutive and Immunomodulatory

Part II Basics of IgG Concentrates

11 Basics of Immunoglobulins as Effector Molecules and Drugs������������ 133

Part III Immune Thrombocytopenia: The First

Part IV Translation from Polyclonal to Monoclonal Antibody Treatment

Paul Imbach is a highly respected pediatric oncologist-hematologist who has

Basel, Switzerland Paul Imbach

1.1 History of New Observations

1980 Pediatric Hematology-Oncology, University Children’s Hospital Berne,

© Springer International Publishing AG, part of Springer Nature 2018 1

1981–1985 During a consultation at the University Children’s Hospital Basel,

CHARACTERISTICS OF TWO STUDY GROUPS

*No significant difference (p=0.337)

36 of 47 (77%) patients randomized to corticosteroids and 39 of 47 (83%) randomized

The percentage of patients with a platelet count of >30x109/l, >100x109/l, or >150x109/l

0.000 0.001 0.008 0.222

0.028 0.023 0.067 0.150

1990 PI transferred to the University Children’s Hospital, Basel, where he contin-

1.2  he Translation of IVIG From ITP to Other Autoimmune

Since the immunopathophysiology of ITP is similar in many other autoimmune and

1.3 The Bridge of Polyclonal and Monoclonal Antibodies

Furthermore the bridge to the engineered, monoclonal antibodies is in the focus of

The description of agammaglobulinemia by Bruton (1952) was a milestone in the

© Springer International Publishing AG, part of Springer Nature 2018 15

2.2 IgG for Replacement: How Much?

• There is a clear pharmacokinetic difference between iv and sc administration.

For further details of replacement therapy, I would like to refer to existing

2.3 Immunomodulation in Autoimmune Diseases

FcR Blockade by IgG

2.4 Immunomodulation in Alloimmune Diseases

Rhesus Hemolytic Disease - Pathogenesis

These can cross the

2.5 Immunomodulation in Inflammatory Diseases

2.6 Expansion to New Indications

Inspired by the fascinating chance to modify immunopathology by IgG as a kind of

Immunomodulation with IgG:

This chapter focuses on practical issues of management. A short summary presents

3.2 X-Linked (Bruton’s) Agammaglobulinemia

3.2.1 Definition and Prevalence

X-linked agammaglobulinemia is an autosomal recessive disorder characterized by

© Springer International Publishing AG, part of Springer Nature 2018 23

The disease is caused by various mutations of BTK (Bruton’s tyrosine kinase)

3.2.3 Clinical Manifestations

Recurrent infections: pneumonia, sinusitis, otitis, later on bronchiectases and gastro-

3.2.5 Differential Diagnosis

Severe combined immunodeficiency (SCID, see 3.3), transient hypogammaglobu-

3.3 Severe Combined Immunodeficiency (SCID)

3.3.1 Definition and Prevalence

3.3.2.1 Different Forms

3.3.3 Clinical Manifestations

Sterile environment, avoidance of live vaccines, avoidance of non-irradiated blood

–– Antibacterial, antifungal, and antiviral treatment of infections

3.4 Hyper-IgM Syndromes

Adrian Newland, CBE, MA, FRCP, FRCPath

1 The Clinical Translation of Intravenous Immunoglobulin

Part I Update of Substitutive and Immunomodulatory

11 Basics of Immunoglobulins as Effector Molecules and Drugs�� 133

Part III Immune Thrombocytopenia: The First

Basel, Switzerland Paul Imbach

1.1 History of New Observations

1.2 he Translation of IVIG From ITP to Other Autoimmune

1.3 The Bridge of Polyclonal and Monoclonal Antibodies

2.2 IgG for Replacement: How Much?

2.3 Immunomodulation in Autoimmune Diseases

2.4 Immunomodulation in Alloimmune Diseases

2.5 Immunomodulation in Inflammatory Diseases

2.6 Expansion to New Indications

3.2 X-Linked (Bruton’s) Agammaglobulinemia

3.2.1 Definition and Prevalence

3.2.3 Clinical Manifestations

3.2.5 Differential Diagnosis

3.3 Severe Combined Immunodeficiency (SCID)

3.3.1 Definition and Prevalence

3.3.2.1 Different Forms

3.3.3 Clinical Manifestations

3.4 Hyper-IgM Syndromes

3.4.2 Clinical Presentation

3.5 Common Variable Immunodeficiency (CVID)

3.5.1 Incidence and Diagnosis

3.5.2 Clinical Manifestations

3.6 Selective Antibody Deficiency

3.6.1 Definition and Diagnosis

3.7 Transient Hypogammaglobulinemia of Infancy

3.7.1 Definition and Diagnosis

3.8 Isolated IgG 1–3 Subclass Deficiency

3.9 elective IgA Deficiency with or without IgG2

3.10 Wiskott-Aldrich Syndrome (WAS)

3.10.2 Clinical Manifestations

3.11 Ataxia Telangiectasia

3.11.2 Clinical Manifestation

3.12 Secondary Immunodeficiency

3.12.2 Specific Aspects

3.12.2.1 CLL and MM

3.12.2.2 Hematopoietic Stem Cell Transplantation HSCT

3.12.2.3 Solid Organ Transplantation

3.12.3 HIV Infection in Children

3.12.4 Preterm Infants

3.12.5 Geriatrics: Immunosenescence

3.13 Other Combined Immunodeficiency

4.2 General Characteristics of Autoimmune Disorders

4.3 eneral Characteristics of IVIG

4.4 linical Categorization of IVIG Indications

4.5.1 A, a* Immune Thrombocytopenia (ITP) (*See Table 4.1)

4.5.2 A, a* Alloimmune Thrombocytopenia (*See Table 4.1)

1. A, a Fetomaternal alloimmunization (See Table 4.1)

4.5.5 B, a, c* Autoimmune Neutropenia (*See Table 4.1)