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Safety Assessment and Dose Selection for
First-in-Human Clinical Trials With
Immunomodulatory Monoclonal Antibodies
PY Muller1 and FR Brennan2
Modulating immune responses with monoclonal antibodies (mAbs) that target immune molecules has become a
promising therapeutic strategy and is under investigation for the treatment of cancer and (auto)-immune diseases.
A major hurdle to the development and early clinical investigation of many immunomodulatory mAbs is the
inherent risk of adverse immune-mediated drug reactions in humans, such as cytokine storms, autoimmunity, and
immunosuppression. Dose selection for first-in-human (FIH) clinical trials involving immunomodulatory mAbs, and
mAbs in general, is based on specifically designed preclinical safety studies, primarily in nonhuman primates (NHPs), and
on mechanistic ex vivo investigations. Dose selection in such trials is challenging for a number of reasons related to safety.
In this context, safety-relevant differences between NHP and human immune systems, species selection/qualification
and preclinical study design considerations, the receptor occupancy model and its calculation, the minimal anticipated
biological effect level (MABEL) and its use in the selection of a safe starting dose in humans, microdosing and the impact
of immunogenicity on safety assessment of mAbs, and safety-relevant formulation properties of therapeutic mAbs are
critically reviewed. In addition, the current regulatory requirements are presented and discussed to demonstrate how the
TeGenero TGN1412 case is leading to increased regulatory scrutiny regarding dose selection for FIH clinical trials.
Immunomodulatory monoclonal antibodies (mAbs) are
biopharmaceuticals that either enhance or suppress immune
responses. With their ability to enhance the immune response
against tumor cells, they have an unprecedented potential for
use in the treatment of cancer.1,2 Using mAbs to stimulate the
immune response against cancer cells employs an indirect
mode of action, achieved primarily by either blocking inhibitory receptors such as cytotoxic T-lymphocyte-associated
protein 4 or triggering/activating costimulatory receptors
such as 4-1BB, CD40, or CD28. The underlying pharmacodynamic (PD) effects are mediated primarily by T cells or natural
killer cells. Conversely, the immunosuppressive properties of
immunomodulatory mAbs are explored for possible use in the
treatment of autoimmune diseases and suppression of transplant rejection3 by suppression of immune-function cells, by
prevention of their homing to lymphoid organs and inflammatory sites, or by induction of anergy or depletion of these
cells. An alternative mode of action involves blocking of soluble
targets, such as cytokines, by mAb-mediated sequestration. For
a general overview of licensed mAbs, their isotypes, targets,
indications for use, and adverse reactions, the recent compilation by Tabrizi and Roskos is recommended.4
In general, mAbs (including immunomodulatory mAbs) have
proved to be safe and (in many cases) effective pharmaceuticals,
whose toxicity is usually related to exaggerated pharmacology
which can, in many cases, be predicted. However, the recent
well-publicized adverse events observed with an immunomodulatory anti-CD28 superagonist mAb (TGN1412, intended to
induce antitumor T-cell response in patients with B-cell chronic
lymphocytic leukemia) in a first-in-human (FIH) clinical trial
in the United Kingdom5 has highlighted how, on rare occasions,
mAbs can be highly toxic. It is for this reason that preclinical
safety assessment programs for mAbs, together with their dose
selection for first clinical entry, will be scrutinized more than
ever by the regulatory authorities in the years to come. Dose
selection for FIH clinical trials (usually of double-blinded, placebo-controlled, single ascending dose design) needs to carefully balance safety of clinical trial subjects (particularly at first
clinical dose administered) and efficient dose escalation to reach
therapeutically active dose ranges. The latter requirement is an
1Novartis Institutes for BioMedical Research, Basel, Switzerland; 2Novartis Horsham Research Centre, Horsham, UK. Correspondence: PY Muller
([email protected])
Received 28 October 2008; accepted 2 December 2008; advance online publication 28 January 2009. doi:10.1038/clpt.2008.273
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Table 1 Guidance documents supporting first-in-human trials with mAbs in general
Guideline title
Authority
Year
Ref.
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use
FDA/CBER
1997
12
Guideline on Production and Quality Control of Monoclonal Antibodies
EU
1994
13
S6: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals
ICH
1997a
7
S8: Immunotoxicity Studies for Human Pharmaceuticals
ICH
2005
11
S9: Nonclinical Evaluation for Anticancer Pharmaceuticals (draft, step 2)
ICH
2008
18
Nonclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals
FDA/CDER
Tissue crossreactivity
In vivo preclinical safety
b
Microdosing, eIND, and eCTA
Position Paper on Non-Clinical Safety Studies to Support Clinical Trials With a Single Microdose
EMEA/CHMP
2004
43
Exploratory IND studies
FDA/CDER
2006
42
Guidance to the Conduct of Exploratory Trials in Belgium
FAMHP
2008
41
Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy
Volunteers
FDA/CDER
2005
16
Guideline on Strategies to Identify and Mitigate Risks for First-in-Human Clinical Trials With Investigational
Medicinal Products
EMEA/CHMP
2007
17
EMEA/CHMP
2007
46
First-in-human dosing and MABEL
Immunogenicity (preclinical and clinical)
Guideline on Immunogenicity Assessment of Biotechnology-Derived Therapeutic Proteins
CBER, Center for Biologics Evaluation and Research; CDER, Center for Drug Evaluation and Research; CHMP, Committee for Medicinal Products for Human Use; EMEA, European
Medicines Agency; EU, European Union; FAMHP, Federal Agency for Medicines and Health Products; FDA, US Food and Drug Administration; ICH, International Conference on
Harmonisation; IND, investigational new drug; MABEL, minimal anticipated biological effect level.
aIn revision. bDraft guidance, not yet officially released.
ethical obligation for (open-label) trials involving patients rather
than healthy volunteers, as is usually the case in oncology.
This review aims to present a comprehensive overview of the
scientific and regulatory considerations for safety assessment of
immunomodulatory mAbs, particularly those targeting cell surface immune receptors, leading to dose selection for first clinical
entry. It should assist clinical investigators and sponsors to critically and independently assess clinical protocols and investigators’ brochures for safety and dose selection of mAb therapeutics
to be administered in FIH trials. A readily applicable representation for dose estimations aiming at low receptor occupancy
(RO) following the minimal anticipated biological effect level
(MABEL) approach is presented. TeGenero’s TGN1412 is used
as an example, both to support this receptor saturation approach
and to highlight how this case is leading to increased regulatory scrutiny and rapidly changing the regulatory framework
(outlined in Table 1).
Preclinical Safety Assessment For
Immunomodulatory Mabs
A major hurdle in the development and early clinical investigation of immunomodulatory mAbs with agonistic, immuneactivating mode of action is the relatively high inherent risk
of adverse drug reactions in humans, such as systemic inflammatory reactions or manifestation of (generally reversible)
autoimmunity.6 Risk prediction of such adverse reactions and
dose selection for FIH clinical trials is based on preclinical safety
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assessment in at least one pharmacologically relevant animal
species.7 The selection of a relevant animal model (i.e., one in
which the mAb binds to the target and elicits the same pharmacological effect as that expected in humans) and data interpretation of toxicity studies in such a model are paramount and
need to be considered and justified on a case-by-case basis. It
is acceptable to conduct studies in only one species of animal
if only one relevant model can be identified, which is often the
case for mAbs. Animal studies should be supported by ex vivo
investigations using human and animal cells and tissues to determine the relative potency of the mAb in humans and the chosen
animal model, to characterize the pharmacological activity in
humans, and to examine specific aspects of mAb safety (discussed later).7 PD end points, if available, should be routinely
assessed in toxicity studies to demonstrate pharmacological
activity of the mAb in vivo in the test model. Generally, repeat
dose toxicity studies using dose levels and exposures representing multiples of the starting dose and highest dose in humans,
with a dosing duration of 4, or sometimes 13 weeks (depending on the duration of exposure in the FIH study) followed by
an exposure-free recovery period of 4–8 weeks (the duration
depending on the predicted duration of exposure and pharmacological activity; refer to “Pharmacokinetic Considerations”)
are used for generating data to support human entry. The dosing
duration in FIH studies for non-life-threatening indications is
usually limited to the dosing duration covered in the animal
­studies.7 However, for life-threatening indications, such as in
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oncology, treatment duration in FIH studies may considerably
exceed preclinical coverage in animal studies. Most of these considerations are applicable to mAbs in general.
The high species specificity of the sequence/structure of most
immune receptors targeted by mAbs makes the selection of an
appropriate animal model challenging. Given its genetic and
pharmacological similarity to humans, the nonhuman primate
(NHP) is the most commonly selected animal model for safety
assessment of mAbs. For practicability/availability reasons the
cynomolgus monkey (Macaca fascicularis, belonging to the family of Old World monkeys—Cercopithecidae) is the preferred
NHP species. Occasionally marmosets (New World monkeys)
and rhesus macaques are used. NHPs are normally the most
relevant model for safety testing of mAbs, because the pharmacological activity in monkeys often resembles that in humans
more strongly than that in lower species, such as dogs, rabbits,
and rodents. In addition, the NHP immune system is generally more akin to that in humans than to that in lower species.
Furthermore, human/humanized mAbs are likely to be less
immunogenic following chronic dosing in NHPs than in lower
species. In some rare cases, the only naturally occurring species
in which certain very selective mAbs exhibit pharmacological
activity is our closest evolutionary relative, the chimpanzee (Pan
troglodytes, belonging to the family of great apes, Hominidae).
However, toxicity studies involving terminal investigations
are justified only in exceptional circumstances in this species,
because of ethical considerations.
Apart from the NHP, the relevance of a second species,
e.g., rodent, needs to be assessed. In relatively infrequent
instances when the mAb shows target binding and pharmacological activity in a rodent, most health authorities expect the
rodent to be used as the second species for testing.7 In such cases,
short-term toxicity studies (e.g., up to 4 weeks) are often performed in rodents. The immunogenicity of human/­humanized
mAbs often results in the generation of antidrug antibodies
(ADAs) that facilitate mAb clearance and/or neutralize the pharmacological action, thereby restricting the duration of toxicity
studies in rodents (refer to “Immunogenicity Considerations”).
Toxicity studies in rodents and NHPs will not be informative
and should therefore be ended when ADAs neutralize/reduce
mAb exposure to levels that are not biologically meaningful or
exert toxicities that are unlikely to be relevant to humans given
the relative immunogenicity across species. However, if pharmacologically active exposure can be maintained in rodents,
studies of longer duration would be expected.
If no pharmacologically relevant animal model is available,
the use of a surrogate mouse mAb (recognizing the mouse
homolog of the human target)8 or testing of the human mAb in
human target transgenic mice9 could be considered as options,
depending on the specific pharmacological/toxicological end
points intended to be assessed and whether they are likely to be
predictive of the outcome in humans in these alternative toxicology models. What is common to both these approaches is
the technical challenge of demonstrating the pharmacological
relevance of the model in assessing safety in humans. Differences
are potentially likely between rodents and humans with respect
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to target ortholog/human transgene expression, turnover, and
signal transduction, and this makes the extrapolation to humans
of any animal-study findings very difficult. In addition, mAbs
against different epitopes on the same target may have different
toxicological profiles, thereby challenging the relevance of the
surrogate approach, at least for some targets. In the case of a
surrogate mAb, a second manufacturing process needs to be
developed in order to produce a high-quality, well-characterized
surrogate mAb for good laboratory practice toxicology studies,
whereas testing of the human mAb in transgenic mice gives rise
to problems of potential immunogenicity, resulting in restriction
of the dosing duration. However, if the NHP is the only relevant
naturally occurring animal model, neither surrogate mAb testing nor transgenic mice models expressing the human target
are routinely required as a second species for study (authors’
experience).
For mAbs with low general toxicity, the establishment of a
maximum tolerated dose, as is done for new chemical entities, is
generally not feasible in toxicity studies because doses can be so
high (i.e., g/kg range) that potential effects might be confounded
by protein overload. Instead, animal-study doses and/or exposures that provide an adequate number of multiples ­(typically
≥10×) relative to the anticipated clinical dose or exposure should
be used as the (highest) doses with the aim of complete target saturation for the duration of the study. The frequency of
dosing may be greater than the one intended to be used in the
clinic, so as to compensate for potential differences in clearance
and/or pharmacology. For mAbs with certain modes of action
(refer to “The MABEL Approach to Determining Safe Starting
Dose”), studying the intended clinical dose(s) as the (lowest)
dose in the toxicity study might be warranted. Dose selection
needs to be based on interspecies scaling, taking into account
species-specific relative affinity/potency of a mAb, which is
often lower in preclinical animal models than in humans. For
immunomodulatory mAbs with agonistic/antagonistic mode of
action, the doses in animal toxicity studies should be increased
accordingly in relation to the intended clinical doses, in order to
achieve complete activation/inhibition of the target. For immunomodulatory mAbs with agonistic mode of action, careful
investigation of potential species-specific differences in effector function and/or signal transduction (refer to “The MABEL
Approach to Determining Safe Starting Dose”) is highly recommended, and should be taken into account for dose selection in
toxicity studies.
For mAbs with an immunomodulatory mode of action,
extended histopathology of lymphoid organs10 and immunophenotyping of lymphocytes is recommended as part of toxicity
studies. Apart from the intended 1° immunopharmacological
effects (PD end points), emphasis should be placed on detection of potential pharmacologically unintended and irreversible
changes in immune parameters and/or overt immunotoxicity
before first human entry. The need for screening of further
immune-function-related end points (T-cell-dependent antibody responses, specific immune cell function assays) depends
on the mode of action of the mAb and the signals observed.
A tiered approach similar to the one commonly performed
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for immunotoxicity assessment of new chemical entities at
later development stages11 should be performed preclinically
to support FIH studies involving immunomodulatory mAbs.
Furthermore, immune-function biomarkers may be identified
for use in detecting signs of potential immunotoxicity in ­clinical
trials.
Tissue crossreactivity (TCR) studies (in which binding of the
mAb to human tissues and tissues from the chosen toxicology
species is assessed) are performed both to identify the potential
for mAb binding to tissues in humans and to confirm the relevance of the toxicology species (in which the mAb has a similar
binding profile on human and animal tissues). TCR data also
aid in the interpretation of any potential pathology findings in
toxicity studies and in determining whether the toxicity findings
in animals are relevant to human safety. Specific risks for immunotoxicity, reproductive toxicity, and adverse safety pharmacology effects may be flagged on the basis of TCR findings. Usually
a full tissue list (32–35 fresh snap-frozen tissues) from three
human and two animal donors is tested for TCR.12,13 Special
absorption, distribution, metabolism, and elimination studies
using radiolabeled mAbs are usually not informative for mAbs,
because cleavage by proteases makes data interpretation difficult.
These kinds of studies are therefore not required for mAbs (the
TCR assesses potential distribution in humans).
In vitro cardiac safety pharmacology assays (hERG channel
activity) are not considered necessary for assessment of QT
interval prolongation risk for mAbs as part of the preclinical
safety strategy.14 mAbs therapeutics have very low potential to
interact with intracellular (unlike low-molecular-weight compounds) or extracellular domains of the hERG channel and are
therefore highly unlikely to inhibit hERG channel activity based
on their targeted, specific binding properties. Furthermore,
mAbs do not gain access to the cytosol of intact cells, because
they reside in the endosomal compartment. Similarly, given
the very low potential for direct DNA interaction, genotoxicity
­studies are usually not required for mAbs.7
Potential in vivo cardiovascular effects can be evaluated by
standard electrocardiograms and blood pressure measurements in restrained animals within NHP toxicology studies
or in conscious, freely moving NHPs fitted with noninvasive
telemetry jackets. Along with cardiovascular parameters, potential cytokine release should be measured (preferably 2–6 h after
the dose) wherever there is any concern relating to the mode
of action of the mAb. A dedicated NHP cardiovascular safety
pharmacology study by telemetry in conscious, freely moving
animals to measure arterial blood pressure, heart rate, and ECGs
may be warranted on the basis of specific study findings, if there
is a mechanistic basis for a potential cardiovascular effect or if
the mAb binds to tissues such as heart, lung, or brain in TCR
studies (particularly for mAbs in the immunoglobulin G1 (IgG1)
format due to Fc effector function).14 On the other hand, if the
TCR studies show the absence of binding of mAb to major organ
systems, separate safety pharmacology studies are not considered to be required.
There are other important safety end points that are required
to be assessed later in the process of clinical development and
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prior to registration of mAbs, and these are not covered in detail
in this review. These end points depend on the duration of treatment, the target population, and the mechanism of action; they
include chronic toxicity (usually 26–39-week studies), reproductive toxicity (e.g., a combined embryo-fetal development and
peri-/postnatal development study; there are usually no dedicated fertility studies, but fertility parameters can be assessed
in chronic toxicology studies in sexually mature animals), and
carcinogenicity (standard 2-year studies in rodents, rarely performed because of immunogenicity and lack of pharmacology
in rodents).
Potential adverse effects may also be predicted depending on the distribution and function of cells expressing the
­t arget receptor and on its species specificity, under both
healthy and pathophysiological conditions. However, despite
very similar target distribution, target expression (TE), and
­target sequence in NHPs and humans, downstream biological
effects may differ considerably following target modulation,
as evidenced by the adverse reactions reported during the
FIH trial with TGN1412, an IgG4-based CD28 superagonist
that activates T cells.5 Therefore, preclinical safety studies for
immunomodulatory mAbs should be supplemented with science-based, mechanistic, ex vivo data using human cells and
a range of mAb concentrations in order to assess the shape of
the dose–response curve. Such experimental data may provide
essential information for dose selection in FIH trials based
on the MABEL, by incorporating all available in vitro and
in vivo data.15
The MABEL approach is now routinely supplementing the
classic concept of dose selection in FIH studies based solely on
the “no observed adverse effect level” (NOAEL) in preclinical
toxicity studies combined with appropriate interspecies scaling,
i.e., the human equivalent dose (HED).16 For biopharmaceuticals >100 kDa that are administered intravenously, and for
which distribution is largely confined to the vascular space, the
HED should be calculated on the basis of body-weight (mg/
kg) scaling.16 For biopharmaceuticals <100 kDa, body surface
area scaling (mg/m2) may be considered (e.g., for mAb fragments such as Fabs or nanobodies). Ultimately, the dose levels
calculated by the NOAEL and MABEL approaches are weighted
by appropriately justified safety factors (usually ≥10) based on
potential risk/hazard, and then compared against each other
in order to select the lower value as the actual starting dose in
humans.
The general aspects of risk mitigation relating to the organizational conduct of FIH trials with higher-risk medicinal
products, including dosing sequence, rescue medication, and
the ­qualifications of the investigators, are discussed in terms
of regulatory issues in a recently released European Medicines
Agency (EMEA) guideline.17
Special Considerations For Safety Assessment
In Oncology Indications
The early development of oncology drugs most often involves
first-in-human clinical trials in cancer patients with latestage or advanced disease with limited therapeutic options.
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Therefore, the study type and timing and the flexibility called
for in designing nonclinical safety studies of anticancer pharmaceuticals can differ from those for other pharmaceuticals. A more flexible safety-assessment approach is endorsed
in an only very recently released International Conference
on Harmonisation Guideline, “Nonclinical Evaluation for
Anticancer Pharmaceuticals” (S9, draft, step 2), which applies
equally to low-molecular-weight drugs and biotechnologyderived pharmaceuticals such as mAbs.18
Phase I clinical trials in cancer patients may include dosing
up to a maximum tolerated dose and dose-limiting toxicity.
Therefore, determination of an NOAEL in preclinical toxicology studies is not considered essential to support human entry.
Dose escalation in clinical trials in patients with cancer should
not be limited by the highest dose tested in preclinical studies. However, when a steep dose–response curve is observed
in toxicology studies, or when no preceding marker of toxicity
is available, a slower escalation should be considered. For anticancer mAbs with agonistic mode of action, selection of the
starting dose in first-in-human clinical trials should be considered using the MABEL approach (see “The MABEL Approach
to Determining Safe Starting Dose”).
Safety-Relevant Functional Differences
Among Primate Immune Systems
Despite the fact that NHPs and humans share a close evolutionary relationship, their immune systems have only limited
similarity. This is because of the rapid evolution of the immune
system in the direct ancestors of humans. There are a number
of significant safety-relevant functional differences between
the immune systems of NHPs and humans, and these need to
be considered while assessing the safety of immunomodulatory mAbs.
Compared with the T cells of our closest evolutionary relative,
the chimpanzee (which, however, is not a direct human ancestor), human T cells give much stronger proliferative responses
upon activation via the T-cell receptor. The underlying mechanism involves human-specific loss of T-cell Siglec expression
during the later stages of evolution, subsequent to the last
common ancestor of humans and great apes.19 CD33-related
Siglecs, which are inhibitory signaling molecules expressed
on most immune cells, downregulate cellular activation pathways via cytosolic immunoreceptor tyrosine-based inhibitory
motifs.20 Among human immune cells, T lymphocytes are a
striking exception, expressing few to none of these molecules,
in contrast to those of chimpanzees and other primates such
as cynomolgus macaques. Moreover, several common human
T cell–mediated diseases, such as bronchial asthma, rheumatoid
arthritis, and type 1 diabetes, have not been reported in chimpanzees or other great apes. These human-specific differences
in T-cell activation need to be carefully considered during safety
assessment of immunostimulatory mAbs against T-cell targets
(see later text).
Fc receptors are plasma membrane glycoproteins that bind
to the Fc region of antibodies. Crosslinking of Fc receptors by
Ab-opsonized antigen complexes initiates cellular immune
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responses, including phagocytosis, Ab-dependent cell–mediated
cytotoxicity (ADCC), respiratory burst, release of cytokines and
inflammatory mediators, and antigen presentation.21 For certain anticancer mAbs, ADCC is intended as a direct mediator
against tumor cells. Human FcγRIII, also known as CD16, is
specific for IgG1 and IgG3. In humans, CD16a is expressed on
monocyte subpopulations, macrophages and natural killer cells
whereas the CD16b isoform is exclusively expressed on neutrophils and eosinophils that have been exposed to interferon-γ
(IFN-γ). However, in NHPs only one CD16 gene, homologous to
the human CD16a, is present. In baboon, rhesus, and cynomolgus monkeys, CD16a expression is restricted to natural killer
cells and monocytes.21 These differences in cell-type expression
should be carefully considered for safety evaluation of therapeutic mAbs, particularly if neutrophils and/or eosinophils are
expected to be involved in any kind of human-specific downstream effects.
Regarding IgG4 effector function, it has been shown that
human IgG4, the underlying format of TGN1412, binds FcγRI
relatively weakly but is capable of activating FcγRI-expressing
cells.22 According to an internal report by TeGenero, the F(ab)2
fragment of TGN1412, in contrast to the full Ab, is not capable
of inducing T-cell stimulation (see later). In view of the ongoing
uncertainty regarding species-specific IgG effector functions,
there is an urgent need for quantitative data on systematic crossspecies comparisons of the binding of human IgG subclasses to
Fc receptors in NHPs and in humans.
Most of the therapeutic mAbs that have received approval to
date were developed in the IgG1 format, which is capable of
mediating complement activation (binding to C1q). However,
for immunomodulatory mAbs that bind to membrane-based
targets, the IgG2 or IgG4 format (or silent IgG1 with reduced
Fc effector function) is often used because of the absence of Fc
effector function (to avoid cell depletion). In contrast to IgG1
and IgG3, IgG2 shows little complement activation and IgG4
shows none at all. Apart from complement-dependent cytotoxicity, which is an intended pharmacological effect of certain
mAbs (e.g., direct killing of tumor cells), complement activation
may lead to a number of safety-relevant consequences, including cytokine release, hematological effects, and/or potentially
life-threatening hemodynamic disturbances.23
In spite of the fact that cynomolgus monkey serum indicates
comparably high titers of complement components, hemolytic
activity was shown to be very similar in the sera of cynomolgus
monkeys and humans.24 This finding is supportive of comparable complement potency in cynomolgus monkeys and humans.
However, mechanistic differences in terms of (cross-)speciesspecific IgG/Fc-mediated complement activation,25 particularly related to IgG aggregation status, cannot be excluded and
deserve further investigation.
Infusion Reactions: Hypersensitivity
Reactions Vs. Cytokine Storms
Acute infusion reactions upon administration of mAbs (black
box warning for several approved mAbs) can be true hypersensitivity reactions, namely, IgE-mediated type I hypersensitivity
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reactions (anaphylactic reactions), or anaphylactoid reactions
not mediated by IgE.26 True anaphylactic reactions usually do
not occur upon initial infusion and require a certain sensitization. In contrast, the pathophysiology of anaphylactoid reactions
appears to be secondary to the release of cytokines consequent to
mAb binding to circulating antigen-expressing cells. However,
anaphylactoid reactions are not equivalent to so-called cytokine
storms (see later text). The clinical manifestations of anaphylactic
and anaphylactoid reactions overlap, and both may lead to lifethreatening conditions involving the cardiovascular, respiratory,
central nervous, gastrointestinal, and cutaneous systems. The predictive capacity of animal models with respect to these reactions
is at best very limited, and the occurrence of infusion reactions
in animals should not be extrapolated to humans. In humans,
the management of anaphylactic and anaphylactoid reactions
involves immediate administration of epinephrine, vasopressors, bronchodilators, corticosteroids, and/or antihistamines.26
If untreated, these reactions may be fatal. Therefore, in managing
severe infusion-related reactions, it is crucial that risks are understood and early signs and symptoms are well recognized.
Pleiotropic cytokines or mAb drugs that induce activation of
a large population of (mainly) T cells or natural killer cells have
the potential to initiate a cascade of systemic release of cytokines.
This has been termed a “cytokine storm” and is characterized by
the massive release of both proinflammatory (tumor necrosis
factor–α, IFN-γ, interleukin-1 (IL-1), IL-6) and anti-inflammatory (IL-10) cytokines.27 A cytokine storm is a potentially
fatal systemic immune reaction consisting of a positive feedback
loop between cytokines and recruitment of immune effector
cells. The immediate clinical manifestations are characterized by
life-threatening systemic inflammatory reactions that may lead
to rapid deterioration of cardiopulmonary, renal, and hepatic
functions.5 Furthermore, tissue injury may occur as a result of
cytokines activating vascular endothelial cells, and this in turn
may result in vascular inflammation/damage and may promote
intravascular coagulation and peripheral ischemia.27 Given
the differences in T-cell activation between humans and NHPs
(discussed in the previous section), T-cell-mediated cytokine
storms are hard to predict on the basis of animal models (cf. the
TeGenero case). In humans, the management of cytokine storms
involves administration of corticosteroids and anti-IL-2 receptor
antibodies combined with agents for stabilization of cardiopulmonary parameters if required.5
used for assessment of safe human starting doses and dose escalation of mAbs for which potential adverse reactions are thought
to be predominantly mediated by on-target effects (exaggerated
pharmacological response).
In Figure 2, the antibody dose leading to an RO of 10% is
calculated based on Eq. 3/4 in Figure 1 for a variety of TE levels
ranging from 0.1 to 10 nmol/l. This plot can be readily used to
determine dose levels leading to 10% RO (and/or RO <10% by
linear reduction of the dose; see below).
Eq. 1: K D =
[ Ab free ] ⋅ [ Ta free ] ( Ab tot − Ab o Ta ) ⋅ ( Ta tot − Ab o Ta )
=
[ Ab o Ta ]
Ab o Ta
Eq. 2: RO =
Ab o Ta
Ta tot
Eq. 3 : Ab tot =
Eq. 4 : Ab tot
Abmolar dose
Vinitial,plasma
⇒ Ab dose = Ab tot ⋅ MWAb ⋅V
Vinitial,plasma
K

RO ⋅ Ta tot ⋅  RO − 1 − D
Ta tot 

=
RO − 1
Figure 1 Equations 1–4. Receptor occupancy (RO) for monoclonal
antibody/target interaction and calculation of dose. Under equilibrium, the
binding of an antibody (Ab) to its target (Ta), leading to the formation of an
Ab–target complex (Ab ° Ta), is expressed by the mass–action law outlined
in Eq. 1, whereby KD represents the dissociation constant. In Eq. 2, RO is
expressed as the fraction of the Ab–target complex relative to total target
expression, TE (Tatot). In Eq. 3, the dose (Abdose) of the Ab is expressed by
total Ab concentration (Abtot) multiplied by its molecular weight (MWAb;
assumed to be 150 kDa) and the initial plasma distribution volume after
intravenous administration (Vinitial,plasma; assumed to be 0.036 l/kg). By
combining Eqs. 1 and 2, the Abtot value can be expressed as shown in Eq. 4.
100.00
10.00
10 nmol/l TE
Dose (µg/kg)
state
TGN1412
1.00
1 nmol/l TE
0.10
0.1 nmol/l TE
The Ro Model
For blood-based immune receptors with known quantitative TE,
the RO can be calculated as a function of the mAb administered.
Complete, i.e., 100%, RO is expected to lead to virtually maximum PD effect of a certain mAb, with the duration of saturation
being dependent on the actual dose administered. The main
parameters that influence doses as calculated by the RO model
are outlined in Eqs. 1–4 in Figure 1. The concept behind this
model is to predict and/or support a safe human starting dose
that leads to a very low RO by the mAb administered (typically
<10%), thereby giving rise to only very minor or virtually no PD
effects mediated by the target. The RO model should therefore be
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0.01
0.001
0.01
1
0.1
KD (nmol/l)
10
100
Figure 2 Graphical representation of monoclonal antibody dose leading
to 10% receptor occupancy (RO). The antibody dose leading to an RO of
10% is calculated based on Eqs. 3 and 4 in Figure 1 as a function of the
dissociation constant (KD) for total target expression ranging from 0.1 nmol/l
(red line) through 1 nmol/l (blue line) to 10 nmol/l (green line) in 10 scaling
steps each. Molecular weight is assumed to be 150 kDa; initial plasma
distribution volume is assumed to be 0.036 l/kg. The TGN1412 dose leading
to a predicted RO of 10% in humans is depicted.
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Based on the characteristics of Eq. 3 in Figure 1, changes in
molecular weight or initial plasma distribution volume after
intravenous administration linearly translate into the respective changes in dose; e.g., a 2× lower molecular weight (75 kDa;
e.g., antibody fragments) would result in a 2× lower dose, and a
2× higher initial plasma distribution volume after intravenous
administration (0.72 l/kg) would result in a 2× higher dose.
However, only at low RO levels (<10%) would changes in intended
RO linearly translate into the respective changes in dose; e.g., a
10× lower RO (1%) would result in a 10× lower dose. Conversely,
at higher RO levels, particularly toward the saturation range, there
is considerable nonlinearity between RO and dose (based on the
characteristics of Eq. 4 in Figure 1). This nonlinearity needs to
be taken into account for finding dose-escalation strategies and
should be calculated using the equation in Figure 3.
For medium- and lower-affinity mAbs (i.e., dissociation constant (KD) >10–20 nmol/l), TE has only a very limited influence
on the dose that leads to 10% RO (upper right area in the plot).
Conversely, for high-affinity mAbs (i.e., KD <1 nmol/l), the dose
that leads to 10% RO shows a proportional relationship with TE,
particularly when the TE is relatively high (the area to the upper
left in the plot with function lines parallel to the abscissa). This
relationship needs to be carefully considered while determining
dose selection of mAbs against targets with respect to which the
expression is modulated by the underlying disease. In such cases,
the use of simplified models for calculation of RO without consideration of TE should be discouraged. For high-affinity mAbs,
the actual affinity (sometimes difficult to determine accurately)
has virtually no influence on dose, particularly in the case of
high TE (the area to the left in the plot with function lines parallel to the abscissa).
Alternatively, RO can be calculated for any combination of KD,
total target expression (i.e., TE) and total antibody concentration
by combining Eqs. 1 and 2 in Figure 1 and subsequently solving
the quadratic equation (resulting in the equation in Figure 3),
whereby total antibody concentration is calculated as a function of the Ab dose administered (based on Eq. 3 in Figure 1).
Such a calculation of RO should be routinely performed with
respect to every dose-escalation step in FIH trials. Examples for
calculation (with reference to the TGN1412 case) are shown in
the respective section.
The Mabel Approach To Determining
Safe Starting Dose
For certain risk profiles of therapeutic mAbs, the EMEA recommends calculation of safe human starting dose on the basis of
the MABEL by incorporating all available in vitro and in vivo
RO =
KD + Ab tot + Ta tot −
(− KD − Ab tot − Ta tot ) 2 − 4 ⋅ Ab tot ⋅ Ta tot
2 ⋅ Ta tot
Figure 3 Equation 5. Receptor occupancy (RO) as a function of dissociation
constant (KD), total target expression (Tatot), and total monoclonal antibody
(mAb) concentration. By using this equation, RO can be calculated for any
combination of KD, Tatot, and total antibody concentration (Abtot), where
Abtot is calculated as a function of the mAb dose administered (based on Eq.
3 in Figure 1).
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data.17,28 The calculation of starting dose should utilize all relevant information and follow a weight-of-evidence approach.
Factors to be taken into account include the novelty of the agent
and its mechanism of action, the degree of species specificity of
the mAb and the target distribution, the steepness of the dose–
response curves of biological effects in human and animal cells,
dose–response data from in vivo animal studies wherein relevance to humans has been validated, the calculation of RO vs.
concentration, and the calculated exposure of targets or target
cells in humans in vivo. The species specificity of the mAb/­target
interaction should address and quantitatively compare affinity
and potency, as well as signal transduction effects consequent
to binding, such as duration of pharmacological action.29
Furthermore, the pharmacological impact of potential genetic
polymorphisms of the target in preclinical safety models and in
humans should be assessed.
The EMEA recommends the MABEL approach in the investigation of medicinal products that are associated with increased
risk with respect to (i) mode of action, (ii) nature of the target,
and (iii) relevance of animal species and models. Similarly, with
respect to immunomodulatory mAbs, the MABEL approach is
encouraged in the investigation of medicinal products that fulfill
one of the following criteria:15
1. The mechanism of action is:
• Through acting on a master switch of the immune system (e.g., CD28 or cytotoxic T–lymphocyte-associated
protein 4),
• Through induction or modulation of pleiotropic
cytokines;
2. The type of engineered scaffold is divalent, trifunctional antibodies, etc.; and
3. The target lacks appropriate animal models for safety testing—i.e., the mAb is not pharmacologically active on the
target in animal models (e.g., the epitope or subepitope is
not present in the animal model).
For mAbs fulfilling criteria 1 and/or 2, it might be warranted
to study the doses (scaling based on mg/kg) that will be admini­
stered to humans in relevant animal safety models first. The classic principle—that the highest dose in animal tests that does
not lead to adverse effects (NOAEL) is the most relevant dose
for determination of safety/safety margin—might not apply to
immunomodulatory mAbs, for which lower doses could have a
divergent effect (and even enhanced potency) as compared with
higher doses (with the possibility of U- or bell-shaped dose–
response curves). Furthermore, in such cases, the RO should be
calculated in the animal model for the doses administered.
Species-specific differences in target affinity as well as in
effector functions mediated by Fc domains of mAbs may lead
to differences in avidity, i.e., multivalent binding/crosslinking, capable of ultimately triggering variable downstream biological effects.30,31 Therefore, depending on the downstream
effects mediated upon target binding, an intended RO <10%,
e.g., 1%, may be warranted in FIH starting doses for certain
mAbs, particularly for mAbs with agonistic modes of action.
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Applying arbitrarily chosen safety margins (>>10) to the
MABEL dose is no guarantee that safety is indeed sufficiently
ensured. On the contrary, such an approach could quickly lead
to unrealistically high safety factors and, in turn, to inappropriately low starting doses. It may be more advisable to obtain the
necessary data that could reduce the uncertainty and that would
enable a more accurate extrapolation from animals to humans
than to keep increasing the safety factor.
The major limitation of the MABEL approach is that it cannot
avoid anaphylactic/anaphylactoid and/or other hypersensitivity
reactions.
Pharmacokinetic Considerations
Clearance
Further important factors to be considered are species-specific
TE levels and/or rate of target turnover—potentially mediated
by the underlying disease. In the case of a soluble target, the
binding of the mAb to the target to form a complex will alter the
clearance rate of most targets to one that conforms to the clearance rate of mAb-target complex, i.e., decelerating the clearance
process. As a result, mAb-target complex will accumulate in the
circulation. Conversely, for membrane-bound targets, the binding of the mAb to the target may alter the clearance rate of the
mAb from a relatively slow process to one wherein the mAb
is more rapidly cleared by internalization of the mAb-target
complex (target-mediated disposition). However, the clearance
of mAbs by cell surface targets represents a saturable process (“antigen sink”) (Figure 4). Therefore, clearance rate does
decrease with increasing dose, leading to an overproportional
increase in exposure and half-life, as is seen for trastuzumab32—
particularly when achieving maximum RO. These relations need
to be carefully considered during dose escalation and repeated
dosing in early clinical trials. Knowledge of the expected
­(species-specific) behavior of such processes will take into
consideration the distribution and clearance of the mAb in an
Clearance mainly by targetmediated internalization
Half-life
Clearance mainly
by RES (saturation
of target-mediated
clearance)
Clearance
mAb dose
Figure 4 Clearance of monoclonal antibodies (mAbs) against cell surface
targets. The total clearance represents the sum of (i) specific, targetmediated internalization that is nonlinear and saturable and (ii) nonspecific
clearance that is linear and attributed to the reticuloendothelial system
(RES). Plasma half-life has an inverse relationship with clearance, leading
to relatively long initial half-life (at higher doses or higher plasma
concentrations) and relatively short terminal half-life because of the
prevailing target-mediated clearance (at lower doses or lower plasma
concentrations). At higher doses or higher plasma concentrations, when the
“antigen sink” is saturated, the clearance value approaches the nonspecific
clearance by the RES.
254
appropriate pharmacokinetic (PK)/PD model. Such a model also
needs to take into consideration species-specific FcRn-mediated
IgG recycling and potentially reduced cross-species FcRn-IgG
binding leading to increased clearance.33 In humans, FcRns are
expressed on phagocytic cells of the reticuloendothelial system,
thereby protecting against the rapid clearance of IgG1/2/4 but
also mediating cross-placental and milk transfer. The downregulation of FcRs on cells of monocyte lineage by concomitant
immunomodulatory drugs, such as methotrexate, may reduce
the clearance of mAbs.34 Taking these factors into consideration,
an estimate of the extent and duration of response for a given
species, including humans, can be made. Furthermore, speciesspecific (terminal) clearance rates may need to be taken into
account for selection of the duration of exposure-free off-dose
periods, both in preclinical safety studies (“recovery period”)
and in clinical trials (“washout”) with mAbs. In practice, four to
five (terminal) half-lives followed by a 1-month true exposurefree period prove to be adequate.
Mabel Approach Based On Ro Vs. Hed Based
On Noael: The Tgn1412 Case
For TGN1412, 10% RO would be reached in humans at a dose
of 1.5 µg/kg (assuming a KD of 1.88 nmol/l and a total TE,
total target expression, of 0.648 nmol/l, using Eqs. 3 and 4 in
Figure 1; Figure 2), which is more than 60 times lower than
the actually administered starting dose (0.1 mg/kg) leading to
>90% RO (calculated based on the equation in Figure 3) and
causing life-threatening cytokine release syndrome in healthy
volunteers.5,35
Following this severe adverse drug reaction, a study was carried out on cynomolgus monkeys, which were dosed at 0.1, 0.5,
5.0, or 50 mg/kg using TGN1412 from the same batch that had
been used clinically. All of these dose levels can be assumed to
lead to complete receptor saturation in cynomolgus monkeys.
In contrast to humans, cynomolgus macaques did not experience any adverse reactions at clinical dose level (0.1 mg/kg)
as assessed by general health, blood pressure, heart rate, temperature, hematology, biochemistry, and liver function.28 In
addition, the higher dose levels that had been used in the regulatory toxicity studies of TGN1412 were reconfirmed to be free
of any adverse reactions. The NOAEL was considered to be at
50 mg/kg. Therefore, the HED corresponding to the NOAEL in
cynomolgus monkeys (mg/kg scaling) is more than 30,000 times
higher than the MABEL dose that is predicted to lead to 10%
RO in humans. TGN1412 provides an impressive example of
the divergence between the HED corresponding to the NOAEL
in animal safety studies and the MABEL dose calculated using
the RO model, and it demonstrates that the latter method is the
most suitable for mAbs with such a mode of action.
In rodents, CD28 superagonists induce the preferential expansion of regulatory T cells and can be used for the treatment of
autoimmune diseases. However, in humans, CD28 superagonists, including TGN1412, induce a delayed but extremely
sustained calcium response in naive and memory CD4+ T cells,
leading to pronounced cytokine release, an event not observed
in cynomolgus T lymphocytes.36
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In vitro, cynomolgus lymphocytes did not proliferate when
stimulated with immobilized TGN1412 unless IL-2 was added to
the cultures. In contrast, in in vitro assays using human peripheral blood mononuclear cells, surface-immobilized TGN1412
stimulated the release of cytokines, including tumor necrosis
factor–α, IFN-γ, IL-2, IL-6, and IL-8 and induced a profound
proliferation of human CD4+ lymphocytes. This is in line with
the finding of massive tumor necrosis factor–α and IFN-γ induction in humans5 as compared to the virtual absence of release of
these cytokines in cynomolgus monkeys, even at 500× higher
doses37 (Figure 5). However, borderline induction of IL-5 and
IL-6 was observed in cynomolgus monkeys, which, in retrospect, needs to be considered as a safety-relevant signal indicating cytokine release.
It was recently demonstrated that ex vivo cytokine production of peripheral blood mononuclear cells can be induced
by TGN1412 in the presence of human endothelial cells.38
However, it is unclear whether endothelial cells of human and
NHP origin can similarly crosslink anti-CD28 mAbs and, if
so, whether such crosslinking plays a critical role in cytokine
production in vivo and, further, which Fc receptor is involved.39
TGN1412 did not induce cytokine release or proliferative
responses when presented in the aqueous phase or when
crosslinked in aqueous phase.
These findings clearly demonstrate that the absence of adverse
findings in cynomolgus monkeys, even at exactly the same dose
levels that are planned to be administered clinically, do not
provide sufficient support for extrapolation to arrive at a safe
10,000
50 mg/kg TGN1412
in cynomolgus
monkeys
0.1 mg/kg TGN1412
in humans
1,670
Fold induction
1,000
704
100
18
18
10
1
1
TNF-α
3
2
IFN-γ
1
IL-5
IL-6
IL-2
IL-4
Figure 5 Comparison of cytokine induction profiles of TGN1412 in
nonhuman primates and in humans. Cytokine induction profiles are
compared for TGN1412 doses of 50 mg/kg in cynomolgus monkeys
(as part of the 4-week good laboratory practice toxicity study) and
0.1 mg/kg in humans. Maximum induction was detected 2–24 h postdose
in cynomolgus monkeys (relative to vehicle control) and 4 h postdose in
humans (relative to predose). Furthermore, in humans, massive induction
of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12p70 was detected
4 h postdose (no quantitative data available). The most striking difference
between cynomolgus monkeys and humans in terms of cytokine
induction is the massive induction of tumor necrosis factor–α (TNF-α) and
interferon-γ (IFN-γ) in humans as against the virtual absence of release of
these cytokines in cynomolgus monkeys even at 500× higher TGN1412
dose. However, borderline induction of IL-5 and IL-6 was observed in
cynomolgus monkeys. In retrospect, this needs to be considered a safetyrelevant signal indicating cytokine release.
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starting dose in humans. Therefore, assessment of RO and relative pharmacology between animal models and humans should
be routinely performed, both for preclinical safety studies and
for FIH studies involving mAbs.
Microdosing In Humans
Microdosing is performed to assess pharmacokinetics, distribution, and/or imaging in FIH clinical trials without therapeutic
intent. Such “phase 0” trials require only limited preclinical
safety testing as outlined in the respective guidelines (Table 1).
The concept is to administer a maximum dose of 100 μg of an
investigational drug and less than 1/100th of the dose of the test
substance that is calculated to yield a pharmacological effect in
humans, as determined on the basis of data from animal studies.40 This approach refers mainly to small molecules and is
outlined in guidance documents from the European Medicines
Agency (EMEA), the FDA, and, recently, the Belgian Federal
Agency for Medicinal and Health Products (FAMHP).41–43
However, in the FDA guideline, a maximum dose of 30 nmol
is mentioned for protein products. For mAbs with a molecular
weight of 150 kDa, this corresponds to 4.5 mg, or a dose of 64 μg/
kg (for a 70-kg subject). This dose is considerably beyond 10%
RO, on the basis of KD and TE, for most mAbs, as outlined in
Figure 2. Therefore, for microdosing studies with mAbs, 1/100th
of the predicted pharmacologically active dose in humans is generally the more relevant safety parameter according to currently
accepted safety standards, and it should be determined using the
MABEL approach.
Immunogenicity Considerations
Most mAb therapeutics—even fully humanized mAbs or Fab
fragments—elicit some level of antibody response (antidrug
antibodies, ADAs) against the therapeutic product when administered to humans (and more so when administered to animals).
Immunogenicity can lead to potentially serious side effects,
alterations in PK parameters (mostly increased clearance), and
loss of efficacy. It has been shown in mice that multimeric ADA
immune complexes are rapidly cleared by the reticuloendothelial system—primarily in the liver.44 ADA responses against
mAb therapeutics may be raised as early as after the first exposure; however, such responses are usually much stronger upon
re-exposure. This property needs to be considered in the context of repeated dosing of mAbs in early clinical trials when
the immunogenicity profile of an investigational mAb is poorly
characterized. Shankar et al. suggest a risk-based strategy for
the assessment of ADA responses against biological drugs.45
In accordance with the classification of Shankar et al., ADAs
against immunomodulatory mAbs targeting cell surface receptors are to be considered as “low risk” (antagonistic mode of
action) or “medium risk” (agonistic mode of action; possible
overstimulation of endogenous mechanisms by therapeutic
mAbs crosslinked by ADAs, “superagonist”). Other possible
adverse reactions secondary to ADAs include injection-site
reactions and immune-complex depositions. Furthermore,
longer-term effects of therapeutic mAbs may be masked by
the development of immunogenicity, possibly neutralizing the
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art
­ harmacological/­toxicological action of the mAb (neutralizing
p
antibodies)—both during safety assessment in NHPs and during
clinical investigation in humans.
In preclinical safety studies ADA determination is recommended at baseline, end of dosing, and end of (exposure-free)
recovery. Analysis of potential ADAs in animal studies is
important for the interpretation of PK parameters and is therefore essential in determining and confirming drug exposure
and interpreting toxicity.45 However, the finding of immunogenicity of mAbs in animals, even in NHPs, has only a very
limited capacity to predict the occurrence of ADA in humans.
Conversely, the lack of ADA-related biological consequences
in animal models cannot be interpreted as a predictor of safety
in humans.
For potential low- and medium-risk ADA responses, determinations should be carried out more frequently early in clinical
development and less frequently at later stages, after the immunogenicity profile has been characterized.45 However, during
routine prescription, it is of debatable value to monitor potentially neutralizing ADAs against immunomodulatory mAbs that
target cell surface receptors, because the presence of neutralizing
ADAs will probably not preclude the treatment of patients. The
recently released EMEA draft guideline suggests a three-tiered
analytical approach to monitoring ADAs levels during clinical
trials: (i) screening assays, (ii) confirmation assays, and (iii) neutralization assays.46
It is of paramount importance to implement robust assays
for immunogenicity assessment as early as possible during preclinical development of mAbs therapeutics. Assay design and
assay performance parameters have been thoroughly reviewed
by Mire-Sluis et al.47 The species-independent “bridging ELISA”
format is often favored. In this context, the assessment of assay
interference/competition of ADAs with PK measurements
(ADAs binding to epitopes of mAb drug) and interference/
competition of mAb drug with ADA measurements (“drug-onboard”) is crucial.48 These interferences need to be thoroughly
addressed during method implementation and method validation before FIH testing as well as before initiation of regulatory
toxicology studies.
Considerations Relating To Dosing Formulations
Very low concentrations of mAbs in dosing formulations may be
associated with a considerable proportion of nonlinear surface
adhesion of proteins to plasticware such as tubes and infusion
systems. Such adhesion may result in substantial reduction of the
actually achieved concentrations of dosing formulations. This
leads to considerable overestimation of the true concentration
in dose formulations and, consequently, to overestimation of
the administered dose. Therefore, surface adhesion needs to be
assessed in all stages of the process of development, manufacturing, and analysis of dosing formulations—even during the
preparation of calibrator solutions used in analysis of dosing formulations during preclinical safety studies as well as for release
of the investigational drug product. In particular, this issue
needs to be addressed in low-concentration dosing formulations
administered in FIH testing. Furthermore, the compatibility of
256
the mAb and the formulation with primary packaging materials
and administration systems should be investigated.17
The formulation properties of mAbs that affect immunogenicity, such as formulation and storage conditions, contaminants
and impurities, exposure of neoepitopes on account of denaturation or fragmentation, and aggregation and adjuvant potential
of inactive ingredients, need to be considered as well.45 This is
particularly important given that manufacturing and formulation processes frequently change during early development of
mAbs. Therefore, biological activity/potency as well as preclinical testing in general should be determined using defined reference material that is representative of the batch used clinically
and validated/qualified assays.
Conclusions And Recommendations
This review summarizes safety assessment and dose selection
for FIH clinical trials with immunomodulatory mAbs, based
on the latest scientific and regulatory considerations. The
MABEL approach, including the RO model, is discussed using
TeGenero’s TGN1412 as an example, in order to both support
this receptor saturation approach and highlight how this case
is leading to increased regulatory scrutiny. Safety-relevant differences between human and NHP immune systems are highlighted, demonstrating that safety assessment of mAbs against
T-cell targets is very challenging in NHPs and that NHP safety
data need to be supplemented by data from ex vivo studies using
human cells.
The recent review by Melero et al. provides a comprehensive overview of potential T–cell targets for immunomodulatory mAbs for oncology indications.2 Based on the known
physiological functions of these immune (co-)receptors, the
risk of cytokine release in humans when these (co-)receptors
are activated might be predicted to some extent. However,
­during safety assessment of TGN1412, this was not adequately
performed.
In the case of CD28, three key parameters are very similar,
or even identical, in cynomolgus monkeys and in humans:
the number of CD28 molecules expressed by T cells, 36 the
affinity of TGN1412 to CD28, and the amino acid sequence
of the extracellular CD28–binding domain.49 Furthermore,
the Fcγ receptor binding of TGN1412 is expected to be comparable in cynomolgus monkeys and humans, given the high
degree of Fcγ receptor sequence homology.39 However, none
of these similarities turned out to be predictive for in vivo
potency of TGN1412 on human T–cell activity relative to that
seen in NHPs.
Sequence comparison of CD28 between NHPs and humans
revealed three amino acid exchanges in the corresponding
transmembrane domains.36 These might influence the lateral
interactions of CD28 with other signal-transducing molecules
within the cell membrane and thereby alter the outcome of CD28
stimulation. The calcium signal observed in human T cells after
CD28 activation was associated with the activation of multiple
intracellular signaling pathways, culminating in the rapid de novo
synthesis of high amounts of proinflammatory cytokines, particularly tumor necrosis factor–α and IFN-γ.36 This is in line with the
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massive cytokine induction observed during the TGN1412 trial in
humans5 but not in cynomolgus monkeys37 (Figure 5). The results
reported by Waibler et al.36 clearly demonstrate that ex vivo models that assess cytokine release as an indicator of a cytokine storm
in humans are valuable tools for assessing the relative potencies
of immunostimulatory drugs in NHPs and in humans.
The observation that calcium flux and cytokine secretion
in human cells require crosslinking of TGN141236 raises the
question of which Fcγ receptor–expressing cell types could
have provided TGN1412 crosslinking in vivo and whether such
crosslinking is also observed in NHPs. In case the pharmacological or toxicological mechanism of action of a certain mAb
depends on receptor crosslinking, the possibility of a bell-shaped
dose–response curve (for stoichiometric reasons) needs to be
considered for both NHPs and humans. In this context, it needs
to be emphasized that crosslinking of mAbs or Fab fragments
bound to cell surface receptors can also be mediated by ADAs.45
This is of particular importance under conditions of repeated
dosing in human subjects when ADA immune response becomes
more likely than it is with single dosing.
In vitro and ex vivo assays are required in order to justify
and validate the species selection for preclinical safety testing.
When no relevant, naturally occurring animal model can be
identified for a certain mAb/target, a surrogate antibody or a
transgenic mouse model expressing the human target might
be considered. However, it remains questionable whether the
use of a surrogate antibody or expression of the human target
would be able to adequately mimic the intrinsically high ability
of human T–cell activation19 and to identify a relevant NOAEL
in any animal model. Therefore, the MABEL approach, together
with RO-guided dose selection, needs to be followed in such
cases so as to predict safe starting doses for first clinical entry.
Only recently, an alternative ex vivo flow cytometric method
was developed to experimentally determine the RO that can be
taken to define the MABEL dose for TGN142.50
Despite the differences in sensitivity between NHPs and
humans19 with respect to T-cell stimulation, risk prediction of
cytokine storms in humans needs to account for any observation of even limited cytokine induction in NHPs, such as was
observed with TGN1412.37 In retrospect, such a finding should
be considered a safety-relevant signal. Similarly, changes in
cytokine profiles, even in case very limited, should be critically
reviewed during ongoing FIH trials, particularly from the viewpoint of justifying dose escalation. It is now recognized, based on
a sound body of data, that findings in NHPs are not predictive
for humans with respect to cytokine storms and that probably
no other naturally occurring animal model is able to fully mimic
those events.27 A way forward to prevent the risk of cytokine
storms in humans might be to modify Fc regions or to use Fab
fragments that would minimize effector-cell stimulation associated with cytokine release (if not required for efficacy). However
this strategy has the drawback of being associated with increased
plasma clearance profiles because of impaired or absent FcRnmediated mAb recycling.
In conclusion, safety assessment for FIH clinical trials with
immunomodulatory mAbs calls for a careful assessment of the
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intrinsic safety-relevant properties of the investigational mAb
itself and must also address human-specific target properties
through in vitro, ex vivo, and in vivo investigations. Combining
this information will establish the basis for dose selection using
the MABEL approach.
Conflict Of Interest
The authors declared no conflict of interest.
© 2009 American Society for Clinical Pharmacology and Therapeutics
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