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Bispecific antibodies are designed to engage T-cells locally and specifically in the presence of a
tumor-antigen, often by binding directly to CD3. By binding to CD3 with one arm, and to a
tumor specific antigen, for example, with the other arm, CD3 molecules would cluster on
the T cell, leading to T cell activation independently of the major histocompatibility complex
(MHC) and costimulatory pathways. Blinatumomab was the first T-cell engaging bispecific
therapy approved by the FDA in 2016. Since then, a multitude of bispecific and multivalent
antibodies and antibody fragments have been developed, and are in clinical development,
including dual affinity retargeting antibodies (DARTs) and bi- and tri-specific killer engager
antibodies (BiKEs and TriKEs) (Wei et al., 2022; Allen et al., 2021).
The development of bispecific molecules raises new challenges for nonclinical develop-
ment and safety assessment and necessitates close cross-functional dialog and planning to
ensure a comprehensive safety assessment. The development program for a bispecific anti-
body sh ould consider expanded nonclinical species justification (e.g., to ensure that there is
binding by both antibody arms to both targets in the nonclinical species ensure that there
is binding by both antibody arms to both targets in the nonclinical species), bioanalytical
assay development (e.g., whether it is necessary to characterize antidrug antibodies against
both arms), and toxicology study design, interpretation, and dose selection. Because the
molar relationship between drug and target (that define free vs. bound ratios) can differ
across individuals and species based on target selection (e.g., soluble vs. cell bound), target
regulation (e.g., total target expression, target internalization and production), drug clearance
and bindi ng affinity, the relative distribution of free and bound drug between the two targets
of a bispecific molecule can differ markedly between species and over time. Bispecific T-cell
engaging molecules are designed to activate T cells locally, but these molecules can some-
times form aggregates (sometimes through interactions with antidrug antibodies, ADA) or
FcR-expressing immune cells can cluster the bispecific anti bodies via their Fc-domain. In
those instances, acute systemic immune reactions can arise, called cytokine release syndrome
(CRS) (Van De Vyver et al., 2021).
The BiTE case study
One such platform for which data are emerging are BiTE scFv molecules, which have been
developed in an effort to not only combat minimal residual disease and thereby increase
response rates but also to reduce toxicities associated with systemic immune modulation
(Baeuerle et al., 2009). Cytotoxic T lymphocytes are a central immune component involved
in controlling tumor growth, and their activation toward tumors has been a primary goal
of many immunotherapies (Monjazeb et al., 2012; Mellman et al., 2011; Hoos et al., 2010).
BiTE molecules can transiently tether resting T cells to tumor cells, leading to concomitant
T cell activation and lysis of tumor cells. This T cell activation presumably occurs via high
avidity engagement of CD3 receptors on T cells, which drives formation of a functional im-
mune synapse with proximal target cell in a manner independent of T-cell receptor clonality
normally involved in regulating T cell activation e.g., peptide-MHC or costimulation (Fig. 4.2)
(Offner et al., 2006; Wolf et al., 2005; Dreier et al., 2003; Huppa et al., 2010). Becaus e this
approach to generating a polyclonal T cell activation toward tumor cells circumvents normal
requirements for peptide antigen presentation via MHC molecules, it offers the promise to
overcome MHC down-regulation or suppression of costimulatory signals by tumor cells to
escape immune destruction (Bubenik, 2003 ; Stopeck et al., 2000).
4. Early de-risking strategy for biotherapeutics106
I. Development of biopharmaceuticals
Severe CRS and neurological adverse events are the main reasons for the interruption of
blinatumomab, a BiTE that consists of two scFvs, which can combine with CD19 and CD3
separately, therapy (Tian et al., 2021). CRS is caused by the release of a large number of cy-
tokines and subseque nt systemic inflammation (Frey and Porter, 2016), and is discussed in
more detail in section (Tumor lysis syndrome (TLS), first dose effect and cytokine release syn-
drome induced by mAbs). The clinical manifestations of CRS include high fever, skin rash,
vomiting, and nausea. This problem can be attributed to abnormal acti vation of effector
T cells and macrophages induced by blinatumomab. The neurological adverse events can
be attributed to the redistribution of activated T cells. Activated T cells induced by blinatu-
momab attach to cerebral vessels and move to cerebrospinal fluid (Reusch et al., 2015). Sedi-
mentation of T cells causes microcirculation dysfunction and local ischemia, which ultimately
result in neurological symptoms.
Anti-CD19 BiTEs have a relatively short circulating half-life (w2h)innonhumanpri-
mates and humans, likely due to their small size (w55 kDa) and renal clearance (Schlereth
et al., 2006; Topp et al., 2011; Klinger et al., 2012). Available nonclinical data support a
general model in which T cell activation by BiTEs is dependent on target cell engagement,
suggesting a risk for systemic, polyclonal T cell activation directed toward self (Dreier
et al., 2003). Immune system activation was observed in nonhuman primates (the only
pharmacologically relevant species), treated with 0.1 mg/kg of the anti-CD19 BiTE ( as
five once-weekly intravenous infusions), as evidenced by increased cytokine release
(e.g., IFNg, IL-6, and IL-2), increased soluble CD25 concentration in serum, increased
expression of T cell activation markers (CD69 and MHC class II on CD8
þ
cells), transiently
decreased CD2
þ
lymphocytes, and decreased CD20
þ
B cells. Additional evidence of im-
mune system activation, including mildly elevated body temperature and reversible hy-
potension, was also observed in nonhuman primates. These data confirm observed
adverse effects among B-cell acute lymphoblastic leukemia (ALL) patients with minimal
residual disease or disease persistence after induction and consolidation therapy. These
patients were treated with 15 mg/m
2
/24 h blinatumomab (anti-CD19 BiTE) as a contin-
uous intravenous infusion for 4 weeks (Topp et al., 2011). Noted Grade 3 and 4 adverse
effects in 21 treated patients included lymphopenia (n ¼ 7), leukopenia (n ¼ 2), decreas ed
circulating immunoglobulin (n ¼ 5), infection (e.g., catheter-related, n ¼ 2), increased ALT
(n ¼ 1), and CNS disorders (e.g., seizure, n ¼ 1; headache, n ¼ 1). Many of these effects
may be considered as possibly or likely related to immune system activation and can
be effectively managed in the clinic. Results from clinical studies with this agent highlight
the promising therapeutic potential of such novel approaches to treating canc er (Topp
et al., 2011; Klinger et a l., 2012).
Other recombinant (nonantibody) protein therapeutics
Antibody-based proteins are a subclass of recombinant proteins that have been discussed
separately from other recombinant proteins based on their unique characteristics; the review
that follows also generally applies to nonantibody-based proteins. Platform toxicities for re-
combinant proteins are generally well understood, and the regulatory framework for the
nonclinical development of recombinant proteins has been accepted for over a decade with
General safety considerations related to biotherapeutics in discovery and early development 107
I. Development of biopharmaceuticals
some recent modifications (ICH S6(R1) 2011; CHMP, 2018). Key to the successful nonclinical
safety evaluation of a recombinant protein is the selection of a relevant species and design of
a robust nonclinical program (Bussiere et al., 2009; Baumann, 2009; Lebrec et al., 2010).
Growth factors
A key concern in the nonclinical evaluation of recombinant proteins is immunogenicity,
e.g., the development of antidrug antibodies (ADA). ADA can bind to the biotherapeutic
and alter the dose-effect relationship in the nonclinical study through altering exposure
(e.g., clearing antibodies), pharmacodynamic (PD) activity (i.e., development of neutralizing
ADA), or both. It is now well accepted that the immunogenicity profile in nonclinical species
is not predictive of the immunogenicity response of a therapeutic protein in humans (Ponce
et al., 2009).
The development of neutralizing ADA is of particular concern when the recombinant pro-
tein is similar to critical endogenous proteins (e.g., growth factors) whose inhibition could be
life-threatening. The first biotechnology product to be FDA approved was recombinant insu-
lin for treatment of diabete s. Recombinant insulin was approved in 1982, and up until that
point, insulin had been purified from animal sources. Needless to say, as the immune cells
of the patient reacted to insulin derived from other animals as being foreign, this often led
to development of antiinsulin antibodies, which could lead to faster clearance and loss of
pharmacologic effect. The recombinant insulin was significantly less immunogenic (Fineberg
et al., 1983). Other adverse events caused by immunogenicity toward recombinant growth
factors include cases where patients treated with pegylated recombinant human megakaryo-
cyte growth and development factor (PEG-MGDF) and erythropoietin (EPO; Eprex) devel-
oped ADA that were cross-reactive to their respective endogenous counterparts, leading to
severe thrombocytopenia with PEG-MGDF (Koren et al., 2002; Kuter and Begley, 2002)
and pure red-cell aplasia with Eprex (Casadevall et al., 2002; Brennan et al., 2010).
In addition to the safety of the protein itself, product-related impurities may contribute to
unwanted effects in the nonclinical study, including alternative glycoforms, charge variants,
aggregates, fragments and peptide variants; of these both protein aggregation and alternative
glycoforms can substantially modify the immunogenicity profile of a therapeutic (Rosenberg,
2006; Yu et al., 2012; Alessandri et al., 2012; Millward et al., 2008; Beck et al., 2008).
Several in silico, in vitro and in vivo technologies to predict huma n immunogenicity with a
view to impro ving the safety and efficacy profile of new drugs are emerging (Ponce et al.,
2009). For example, HLA transgenic mice expressing the most common HLA-DR alleles of
the Caucasian population, human adaptive immune system in cord blood cell-transplanted
mice, and new xenotransplantation mouse models such as NOD/SCID/gc
/
or Rag2/
/gc
/
strains have been developed (Hermeling et al., 2005; Gonzalez-Gay et al., 1996;
Ito et al., 2002). Even though these models offer an attractive opportunity to predict liabilities
of human biotherapeutics, human proteins are still immunogenic in these mice. These models
can not only fail due to lack of complete engraftment of all the components of the human im-
mune system but also due to the long time it takes to generate such models. In contrast to the
in vivo approaches, in silico and in vitro prediction methods specifically focus on the contri-
bution of T cells to ADA formation. The advantage of these methods is that they are human-
based and so there is no issue regarding species differences. In addition, these approaches are
relatively easy to apply and their short time course fits well into a research and development
4. Early de-risking strategy for biotherapeutics108
I. Development of biopharmaceuticals
program of a new protein-based therapeutic agent. However, these platforms do not
adequately capture the antigen presentation and development of tolerance, highly unpredict-
able and complex processes, in their entirety and can therefore lead to false prediction of
immunogenicity.
Recombinant cytokines
Recombinant cytokines are generally designed to enhance immunity either by achieving
generalized immunostimulation or stimulating specific immune cells. While these proteins
have improved treatment options for several diseases they have also been associated with
novel toxicities. Toxicities arising from the use of pharmacologic immunostimulation can
be mechanistically interpreted as imbalances in cytokine signaling, due to either cytokinemia
or secondary events resulting in exaggerated production of endogenous cytokines. Therapeu-
tic immunostimulation aims to enhance responses against infection or malignant cells by acti-
vating innate immunity, enhancing adaptive immune responses or overcoming
immunosuppressive signals mediated by the pathogen or tumor microenvironment. Taken
as a whole, evaluation of immunostimulatory proteins in nonclinical models are generally
predictive of PD responses in humans, with a few exceptions such as cytokine storm, first-
dose response, tumor lysis syndrome (given their absence of tumors), and autoimmunity
(Gribble et al., 2007).
Interleukin (IL-12) case study
IL-12, a heterodimeric cytokine produced predominantly by macrophages and other anti-
gen presenting cells, is a key regulator of cell-mediated immunity. Recombinant human IL-12
(rhIL-12) was subsequently shown to increase IFNg production from NK and T cells, enhance
NK/lymphokine-activated killer (LAK) cell cytotoxicity, and promote T-helper cell type 1
(Th1) immune responses both in vitro and in vivo. rhIL-12 was used in clinical trials for its
therapeutic potential as an anticancer and antiinfectious disease agent. These studies demon-
strated that prior single dose administration of a low dose of IL-12 reduced the toxicity of
subsequent, higher doses, and this priming could mitigate lethal toxicities observed in pa-
tients treated with a single dose (Cohen et al., 2002; Leonard et al., 1997). Such a schedule-
dependent effect on pharmacology and toxicity in humans was later confirmed in mice
and nonhuman primates and is attributed to an attenuated pharmacology with repeated
exposure (Clark et al., 2012). Because of this unique schedule-dependent phenomenon, care-
ful attention to dose and schedule is required to assure safe and effective clinical development
of this cytokine.
Interferon (IFN-a) case study
Various forms of recombinant human IFN-a are marketed and used to treat chronic hep-
atitis C virus infection and cancer (Vilcek, 2006). Interferons play a major role in the first steps
of the response to acute viral infections, being mediators in the innate cellular antiviral
response that precedes the adaptive immune response. Specifically, the immunostimulatory
activity of IFN-a includes up-regulation of antiviral gene expression (Smith et al., 2005; Kunzi
and Pitha, 2003; Katze et al., 2002), suppression of cell proliferation (Rosewicz et al., 2004;
Dowding et al., 1993), enhanced cytotoxic activity of macrophages, NK cells and neutrophils
(Bose et al., 2006; Ostensen and Forre, 1988) and inhibition of viral replication (Narayanan
General safety considerations related to biotherapeutics in discovery and early development 109
I. Development of biopharmaceuticals
et al., 1997; Barton et al., 2005). Toxicities associated with chronic dosing in patients are
diverse and include flu-like symptoms, gastrointestinal effects, fatigue, CNS disorders,
including neuropsychiatric abnormalities and sleep disorders. In addition, laboratory abnor-
malities, including anemia, thrombocytopenia and neutropenia, are associated with chronic
treatment and constitute one of the major reasons for treatment discontinuation or dose
adjustment. Despite the emergence of neutralizing antiinterferon antibodies in nonhuman
primates (NHPs), nonclinical safety studies conducted in NHPs were useful in predicting po-
tential human risks of IFN-a therapy.
Evaluation of immunotoxicity
Brennan et al. (2010) extensively reviewed deleterious consequences of unintended im-
mune suppression and activation of immunomodulatory biotherapeutics while demon-
strating their clinical efficacy, based on desirable immunomodulatory effects leading to
intended immunopharmacology. These adverse consequences, considered as immunotoxic
effects, can result from exaggerated or prolonged activity of the biotherapeutic drug binding
to the desired target on intended target cells/mediators, modulating a target with multiple
immune functions (beyond those that are required for therapeutic benefit) or modulating a
target that is also expressed on cells (immune/nonimmune) besides those that are intended
for therapeutic purposes. Such immunotoxic effects can lead to either immunosuppression or
immunostimulation resulting in infection and cancer, autoimmunity, cell/tissue injury,
inflammation, cytokine storm, tumor lysis syndrome or hypersensitivity (Ponce, 2008;
Descotes, 2009).
Brennan et al., (2010) cautions against some effects derived from testing biotherapeutics in
healthy animals used in nonclinical studies. These animals may either express low levels of
the target or may have compensatory/redundant mechanisms that could underestimate
some effects, and it is generally ackno wledged that nonclinical species are not sensitive
models for assessing the risk for adverse effects due to immunomodulation such as CRS.
Notwithstanding these concerns, a range of immunological endpoints can be incorporated
to predict adverse events into dose range-finding (DRF) studies, such as: flow cytometric
assessment of immune subsets, total Ig measurements, serum cytokines, acute phase proteins ,
complement components, ex-vivo neutrophil oxidative burst and phagocytosis measure-
ments, clotting factors, ex vivo leukocyte activation assays, ex vivo leukocyte signal transduc-
tion assays, ex vivo T cell proliferation, receptor occupancy (RO), inflammation at injection
site (local tolerance) and/or other specialized assays .
Specialized assays to monitor for unintended immunotoxicity should be considered based
on target liability assessments related to the primary pharmacology and used in conjunction
with routine clinical and gross/histopathology assessments to identify unanticipated effects.
These cytokine release assays gained more attention after the TGN1412 incident (discu ssed
later in this chapter). One of the first, and the most widely used format of the cytokine release
assays, is the whole blood assay, where whole blood from healthy human donors is stimu-
lated with soluble drug candidate (Wolf et al., 2012). An alternate format consists of isolated
peripheral blood mononuclear cells (PBMC) stimulated with either soluble or immobilized
4. Early de-risking strategy for biotherapeutics110
I. Development of biopharmaceuticals
drug candidate (Vessillier et al., 2015), and the latter in particular was shown to be more sen-
sitive for predicting the hazard for CRS. More advanced assay formats include coculture
PBMC with autologous endothelial cells (Reed et al., 2015) and methods to mimic the circu-
lating blood (Fletcher et al., 2018). The readout from all assays are levels of secreted cytokines,
in particular cytokines known to cause CRS. Global initiatives are ongoing to compare
different assays and to align on which reference compounds to include for comparison (Gri-
maldi et al., 2016; Vessillier et al., 2020).
Immunosuppressive biotherapeutics
Immunomodulatory, mAbs antibodies or Fc-fusion proteins such as muromonab-CD3
(Orthoclone OKT3), alefacept (Amevive, 2002), natalizumab (Tysabri), infliximab (Remicade),
adalimumab, etanercept (Enbrel), efalizumab, abatacept (Orencia), eculizumab (Soliris), ritux-
imab (Rutxan), and alemtuzumab (Campath), that are indicated for the treatment of inflam-
matory/autoimmune diseases or to prevent organ transplant rejection, are often designed to
bind directly to target immune cells or soluble mediators in order to deplete them or suppress
their function (St Clair, 2009; Genovese, 2005; Menter, 2009; Rommer et al., 2008; Isaacs, 2009;
Zareba, 2007). This therapeutically induced immunosuppression can lead to an increased risk
of opportunistic bacterial, fungal, parasitic or chronic viral infection and virally induced can-
cers (St Clair, 2009).
Host resistance assays, T-cell dependent antibody response (TDAR) or other immune func-
tion tests should be considered on a case-by-case basis to assess broad risk for immunosup-
pression with biotherapeutics (Brennan et al., 2010 ). Amongst these, host resistance assays
can be used to determine whether the immune system has become ineff ective in mounting
resistance to invading pathogens, and confirmation of specific immune cell types involved
by decreasing resistance to organisms controlled by those cell types (e.g., increased suscepti-
bility to fungal infections if neutrophil function is compromised) (Brennan et al., 2010; Burle-
son and Burleson, 2008). Despite use of rodent influenza, Candida albicans, and other models
to understand the immunosuppressive effects of biotherapeutics, host resistance assays are
not routinely performed within the industry because their predictive value for humans is un-
proven (Burleson and Burleson, 2007, 2008; Bugelski et al., 2000 ). Therefore, potential
impaired resistance to microbial pathogens is generally addressed in clinical trials and in
the subsequent clinical risk management and pharmacovigilance plans. Brennan et al.,
(2010) also lists a variety of factors that investigators should consider prior to embarking
on rodent host defense studies.
1. Due to overlapping and compensatory immune pathways, effects on immune function
may not result in decreased host resistance unless multiple host resistance models (a
combination of bacterial, viral and tumor models) and immune function tests are
utilized to increase the weight-of-evidence.
2. Caution should be exercised when surrogate biotherapeutics are used in rodent models,
since these studies do not use the clinical version of drug product, differences in
binding properties of the surrogate and downstream signaling events in the animals,
may differ from the human counterpart (pharmacological irrelevance).
Evaluation of immunotoxicity 111
I. Development of biopharmaceuticals
3. Primary endpoint is often mortality, which is insensitive and of debatable utility as a
predictor of immunosuppression.
4. Predictivity of models where susceptibility to infection is dependent both on the degree
of immunosuppression and the number of challenge organisms may not hold true for
humans, where the degree of immunosuppression may be variable in the out-bred
population and the number/nature of challenge organisms cannot be controlled.
Moreover, infection in humans occurs on a background of concomitant medication and
underlying disease (e.g., RA, psoriasis), variables not tested in host resistance models.
5. Available host resistance database is limited to a small number of highly
immunosuppressive drugs and hence the question remains as to whether these models
can detect the effect of mild/moderate immunosuppression on host defense (Brennan
et al., 2010; Luster et al., 1993).
Host resistance assays are rarely performed in NHPs due to lack of qualified models, low
animal numbers, different spectrum of pathogen load, high interanimal variation, lack of spe-
cific pathogen-f ree (SPF) animals and because such studies go against one of the main goals
of early de-risking strategiesdreduction in animal usage (3Rs). However, there has been in
an increased interest in modeling tuberculosis (TB) in NHPs. Macaques are susceptible to
infection with different strains of Mycobacterium tuberculosis, producing the full spectrum of
disease conditions, including latent infection, chronic progressive infection, and acute TB,
depending on the route and dose of infection (Kaushal et al., 2012). In addition, macaques
have been used to monitor immunosuppressive effects of the Fc-fusion proteins alefacept
and abatacept (Amevive, 2002; EMA assessment report). Increased titers of endogenous lym-
phocryptovirus (LCV) were observed after chronic treatment of monkeys with alefacept. LCV
and other tumor-promoting viruses induce polymorphic B cell hyperplasia or plasmacytoid
hyperplasia that could result in lymphoproliferative changes and potentially to lymphoma.
Immunostimulatory biotherapeutics
Immunostimulatory biotherapeutics, especially anticancer agents, (e.g., IL-2, IL-21, rituxi-
mab, alemtuzumab, CTLA-4, GITR, OX40 and CD40) are intended to induce immune activa-
tion, break tolerance and augment antitumor immune responses by activating immune cells
through direct agonism of an immune activation receptor, antagonism of an immune inhib-
itory receptor, or by depleting or inhibiting specific populations of T regulatory cells (Gribble
et al., 2007 ; Cranmer and Hersh, 2007; Valzasina et al., 2005; Khalil and Vonderheide, 2007).
These molecules produce acute first-dose infusion reactions, manifested as cytokine release
syndrome (CRS), tumor lysis syndrome (TLS), or hypersensitivity (pseudoallergic or allergic)
reactions.
Tumor lysis syndrome (TLS), first dose effect and cytokine release syndrome
(CRS) induced by mAbs
Acute CRS, TLS, and infusion reactions are adverse events, not readily detectable in
nonclinical species, associated with some therapeutic antibodies in humans leading to a spec-
trum of clinical signs from nausea, chills and fever to more serious dose limiting hypotension
4. Early de-risking strategy for biotherapeutics112
I. Development of biopharmaceuticals
and tachycardia (Wing, 2008). CRS is an exaggerated systemic immune response involving
release of inflammatory mediators such as cytokines, chemokines, oxygen radicals and coag-
ulation factors (Fig. 4.3). CRS should be differentiated from TLS, a life-threatening metabolic
imbalance caused by the rapid breakdown of primarily liquid tumors such as acute lympho-
cytic leukemia and high-grade non-Hodgkin’s lymphoma (NHL) in response to rituximab
(Fig. 4.4). CRS phenomenon differs from infusion reactions by magnitude and in the elevation
of both proinflammatory (T NFa, IL-1, IL-6) and antiinflammatory (IL-10, IL-1Ra) cytokines.
Infusion reactions have been reported for several mAb drugs, including rituximab (anti-CD20
mAb), muromumab (anti-CD3 mAb) and alemtuzumab (CAMPATH, anti-CD52 mAb).
Albeit different in origin, infusion reactions associated with rituximab, muromumab, and
alemtuzumab encompass multiple adver se events that occur within 30e120 min from start
of the first infusion. These include primary symptoms such as fever and chills/rigors, nausea,
urticaria, fatigue, headache, pruritus, bronchospasm, dyspnea, sensation of tongue or throat
swelling, rhinitis, vomiting, hypotension, sterile meningitis, flushing and pain at disease sites
(Gribble et al., 2007). The incidence of infusion-related events decreased from 70% to 80%
during the first infusion of muromumab to w40% with subsequent infusions, perhaps due
to disappearance of target cells.
TGN1412 case study
TGN1412 was a sup eragonist mAb specific for the T-cell costimulatory molecule CD28 and
induced a life-threatening systemic release of proinflammatory cytokines (CRS), in six healthy
volunteers in 2006 (Suntharalingam et al., 2006). CD28 costimulates T cell responses when
engaged by CD80 and CD86 present on “professional” antigen-presenting cells during anti-
gen recognition. TGN1412 induces particularly strong CD28 signaling, allowing T cell activa-
tion without the need for simultaneous strong TCR engagement (Tacke et al., 1997). Up until
the Phase 1 clinical trial of TGN1412, adverse effects of infusion of agonistic biotherapeutic
(e.g., muromon ab) into man could be predicted with a degree of confidence using standard
assays based on PBMC or whole blood. In this case nonclinical data in rodents, nonhuman
primates and human cells failed to predict CRS. Since this incident, a great deal of attention
has been focused on in vitro methods that could predict severe adverse reactions seen in the
trial’s volunteers.
A number of investigations have targeted the development of a method for detecting the
response to and the mechanism of action of TGN1412 and other mAbs with the potential to
cause CRS in humans (Romer et al., 2011; Dhir et al., 2012; Findlay et al., 2010; Eastwood
et al., 2010; Sandilands et al., 2010; Stebbings et al., 2007; Wolf et al., 2012). At this time, cyto-
kine release assays (CRA) are not designed to define a threshold or exposure level in which
cytokine release may be a concern with administration to humans, but to identify compounds
that may produce severe CRS (Vidal et al., 2010). Recommendations from a European Med-
icines (EMA) Workshop states “that various approaches can be chosen, from simple test sys-
tems to more complex models including co-cultures, or other systems aimed at mimicking as
far as possible the in vivo situation. The state-of-the-science is not adequate at present to
allow specific recommendations regarding which assays/formats are most appropriate for
different types of target and mechanisms” (Vid al et al., 2010
). Furthermore, the HESI
Immuno-safety Technical Committee Cytokine Release Assay Working Group publis hed
that despite a high interlaboratory variability in the levels of cytokines produced in the
Evaluation of immunotoxicity 113
I. Development of biopharmaceuticals
FIGURE 4.3 Reported inducers of CRS. CRS can be induced by direct target cell lysis with consecutive release of
cytokines such as interferon gamma (IFNg) or tumor necrosis factor alpha (TNFa) or by activation of T cells due to
therapeutic stimuli with subsequent cytokine release. These cytokines trigger a chain reaction due to the activation of
innate immune cells such as macrophages and endothelial cells with further cytokine release. Abbreviations: Ang-2:
Angiopoetin 2; CAR: Chimeric antigen receptor; DC: dendritic cell; IFN-g: interferon gamma; MHC-I: major histo-
compatibility complex I; NK cell: natural killer cell; PD-(L)1: Programmed cell death protein (ligand) 1; TCR: T cell
receptor.; TNF-a: Tumor necrosis factor alpha; vWF: von Willebrand factor. Reprinted from Shimabukuro-Vornhagen
et al., (2018).
4. Early de-risking strategy for biotherapeutics114
FIGURE 4.4 Pathogenesis of tumor lysis syndrome, resulting in acute kidney injury. Reprinted from Lupușoru et al.,
(2022).
Evaluation of immunotoxicity 115
I. Development of biopharmaceuticals
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