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to the more recent Mosunetuzumab (CD20/CD3) and Glofitamab (CD20/CD3). These PVBs
target the CD3 antigen on T cells with one arm of the antibody and the tumor antigen with
the other arm which results in T-cell activation (perforin/granzyme B and cytokine secretion)
and subsequent target mediated cell killing. This mechanism can kill both dividing and
nondividing cells in a serial fashion. Within this platform, both full-length antibody and
BiTE classes exist. From a platform perspective, using the body’s own T cells to identify
and kill target tumors cells comes with specific safety-related concerns, especially considering
the high potency of CD3 bispecific antibodies.
Depending on the target antigen being engaged by T cells, a differential safety profile
could result between molecu les sharing this mechanism of action. Case examples will be
reviewed below for different target antigen TDB therapies (Fig. 2.1), highlighting points to
consider for development of this class of molecules. As the data are discussed, it is important
to note the evolution of development strategies over time with the availability of cross-
reactive molecules in humans, rodents, and cynomolgus monkeys.
Case Example 1. EpCAM-targeting TDBs:
Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein that is
involved in cell migration, proliferation, and differentiation (Maetzel et al., 2009; Munz et al.,
2004; Trzpis et al., 2007). EpCAM is overexpressed in the majority of epithelial tumors thus
making it a desirable target for patients with epithelial cancers such as ovarian, gastric, colo-
rectal, pancreatic, breast, lung, and endometrial tumors (Spizzo et al., 2004, 2006; Went et al.,
2004, 2006).
Catumaxomab (EpCAM/CD3 multitargeting full-length antibody)
Catumaxomab (Removab) is a trifunctional IgG2 antibody approved for the treatment of
malignant ascites, a condition found in patients with a variety of epithelial cancers. It is
composed of heavy and light chains from mouse and rat and binds to human EpCAM and
human CD3 receptors. In addition to variable region binding, the Fc region also has binding
affinity for FcgR1 (CD64), FcgRIIA (CD32a), and FcgRIII (CD16).
Catumaxomab is a protein with target-binding properties specific to human antigens;
therefore, standard animal models were considered inappropriate for preclinical testing
due to lack of target antigen binding. Based on this, repeat-dose toxicity studies were not
TABLE 2.1
Company Molecule name Targets Preclinical safety Clinical safety References
Fresenius
BioTech
GmbH,
Munich,
Germany
RevomabÒ
(catumaxumab);
Full length
antibody
EpCAM CD3 • Performed in antigen-
independent species
(rabbit, rat, Guinea pig
and cynomolgus monkey)
• homolog ab tested in
mouse (only CD3-
dependent binding, no
EpCAM binding)
• homolog ab tolerated at
human equivalent doses
Cytokine release
related symptoms
and
gastrointestinal
reactions
Seimetz et al.
(2010)
EMA
Removab,
INN-
catumaxomab
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals36
I. Development of biopharmaceuticals
TABLE 2.1 dcont’d
Company Molecule name Targets Preclinical safety Clinical safety References
Amgen MT110; BiTE
format
EpCAM CD3 • Surrogate ab (muS110)
evaluated in mouse
toxicity studies
• similar EpCAM
binding; 35-fold
lower CD3 binding
to mouse CD3 as MT110
• muS110-induced
cytokine release in
mouse studies
Pyrexia, increased
LFTs*
Amann et al.
(2008, 2009);
Fiedler et al.
(2010)
Kebenko et al.
(2018)
Amgen Blinatumomab
(MT103)
CD19 CD3 Toxicity evaluation
performed in
chimpanzees.
• findings consistent
with cytokine release
Cytokine release
and CNS toxicity
Schlereth
et al. (2006);
Nagorsen and
Baeuerle
(2011)
Jain and
Litzow (2020)
Zhu et al.
(2016)
Amgen Cetuximab-
based EGFR
BiTE (C-BiTE)
EGFR CD3 Repeat dose toxicity
study performed in
cynomolgus monkey.
• cytokine release leading
to premature termination
of animals at higher doses
• Histopathology findings
consistent with EGFR
expression
Clinical data
unavailable
Lutterbuese
et al. (2010)
GenMab:
Janssen
Biotech;
OmniAb
Inc
Teclistamab BCMA-CD3 • NHP relevant species
lack of
pharmacodynamics
in NHP
• Affinity difference
of target antigen in
NHP versus human
• ADA formation
leading to loss of
exposure
CRS, neurologic BLA 761291
Abbvie/
Genmab
Epicoritamab
(GEN3013)
CD20/CD3 • NHP relevant species
with similar target
affinity to human
• S.C administration
leading to less
cytokine secretion
• ADA formation
leading to loss of
exposure
CRS BLA 761324
T celledependent bispecific antibody biotherapeutics 37
I. Development of biopharmaceuticals
conducted. As such, preclinical pharmacology and PK studies utilized in vitro assays and
specific mouse models to assess activity of catumaxomab (Seimetz et al., 2010)(Table 2.1).
In vivo pharmacokinetic/pharmacodynamic (PK/PD) studies utilized a homolog anti-
body (antihuman EpCAM antimouse CD3) to catumaxomab mouse CD3 but lacked bind-
ing to mouse EpCAM. In order to mimic the clinical indication as closely as possible, specific
mouse models were generated where both cancer cells and immune cells are found in the
peritoneal cavity. These studies demonstrated that the homolog antibody was tolerated at hu-
man equivalent doses.
The catumaxomab toxicology program was conducted using several differe nt nonbinding
species (rabbit, rat, guinea pig, and cynomolgus monkey) in single-dose escalation studies.
Results of these studies concluded that there was no acute toxicity or local intolerability at
injection sites at dose s exceeding the human therapeutic dose range (Seimetz et al., 2010).
When considering the toxicology program for catumaxomab, it is difficult to draw conclu-
sions on potential target-related safety liabilities due to the lack of tumor antigen (EpCAM)
and CD3-dependent binding in the nonclinical toxicology species evaluated. Further, when
considering the mouse data using a surrogate antibody binding only CD3þ T cells in the
absence of tumor antigen binding, T celledependent effects could be identified, but the rela-
tionship of targeting T cells and EpCAM is unknown. In this type of a situation, a more con-
servative clinical dosing strategy was implemented where the first-in-human dose was well
below the anticipated maximum tolerated dose (MTD) and dose escalation schema was very
cautious.
Clinical adverse events were attributed to cytokine release related symptoms and as a pre-
caution, doses well below the MTD were recommended for further clinical trials (Seimetz
et al., 2010). In order to enable IV dosing for additional indications, premedication with ste-
roids and antihistamines was necessary at the MTD (Chames and Baty, 2009). In this case
example, although the predictivity of the preclinical program for the clinical program was
limited, occurrence of adverse effects in the clinical setting was managed with appropriate
treatments, thus not limiting the further development of the clinical progra m.
MT110 (EpCAM/CD3 TDB, BiTE format)
MT110 is a single-chain bispecific antibody construct from the BiTE class targeting human
EpCAM and CD3 for the treatment of adenocarcinoma of the lung or gastrointestinal tract
(Amann et al., 2009). MT110 is only cross-reactive to human CD3 and human EpC AM
(with the exception of chimpanzee) and does not cross react with its orthologs in other species
(Amann et al., 2008). Because the chimpanzee is no longer considered an appropriate species
for in vivo studies, a murine surrogate (muS110) was created consisting of a murine specific
single-chain antibody for EpCAM and CD3 antigens.
Binding affinities for EpCAM were determined to be similar between MT110 and muS110;
however, the affinity of muS110 for CD3 was 35-fold higher than that of MT110 for human
CD3 (Amann et al., 2008). Therefore, with this case example, although target antigen binding
was achieved with muS110 in mouse, the differences in binding affinity between MT110 and
muS110 for CD3 make translating findings from the nonclinical toxicology program to poten-
tial clinical safety liabilities very chal lenging.
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals38
I. Development of biopharmaceuticals
In spite of the antigen binding challenges posed by muS110, a series of single-dose mouse
studies were conducted to evaluate a range of muS110 doses. In these studies, the main
toxicity observed was related to cytokine release syndrome. In follow-up 7-day studies
(Amann et al., 2009), with daily dosing from 2 to 50 mg/kg per day, findings were generally
consistent with single-dose studies. However, repeated daily administration of 50 mg/kg
exceeded the MTD and resulted in pronounced transient clinical signs within 8 h after each
dose administration (hypothermia, body weight loss, piloerection, diarrhea) with 30% of an-
imals being euthanized after the third or fourth injections. Similar transient clinical observa-
tions also occurred at lower doses of muS110 but subsided after the fourth dose
administration. This adaptive effect after low-dose administration of muS110 may have
been due to multiple reasons, including elimination of EpCAM þ lymphocytes as well as
blunting of T cellerelated cytokine release at higher doses of the antibody. In addition to
the clinical signs noted in the in vivo mouse studies, systemic and transient release of inflam-
matory cytokines (IL-2, IFNg, TNFa, IL-6, IL-10, and IL-4) were observed with peak levels
being achieved 2 h after injection. Overall cytokine release was consistently reduced with
subsequent doses of muS110, with no further cytokine release detected after the fifth infusion.
In order to further investigate the adaptive response to muS110, well-tolerated doses up to
10 mg/kg per day were administered daily for 7 days, followed by challenge doses of
100e500 mg/kg per day muS110 (Amann et al., 2009). Results from this study concluded
that repeat administration of low doses of muS110 increased the tolerability of subsequent
infusions of much higher, otherwise lethal doses of muS110.
Clinical results from the phase I study show that MT110 main target related AEs were
considered consistent with the MOA (Kebenko et al., 2018 ). Dose-limiting toxicities included
diarrhea and transient increases in liver parameters. These findings were also seen preclini-
cally, but in the case of the diarrhea, the clinical severity was not predicted (Kebenko
et al., 2018). Similar to the preclinical study evaluating adaptive responses to muS110, the
clinical study demonstrated that step dosing (e.g., low-dose MT110 pretreatment) improved
liver enzyme increases.
Considering the clinical data with catumaxomab, preclinical mouse data with muS110, and
clinical data with MT110, the primary safety liabilities that were identified were consistent
with and possibly related to cytokine release and/or on-target effects on tissues. The avail-
able literature for EpCAM-targeting PVBs, catumaxomab and MT110, demonstrates the evo-
lution of nonclinical safety assessment strategies over time. Overcoming the challenges of
antibodies that bind only human target antigens to the creation of mouse surrogates that
can bind both target antigens demonstrates the complexity of developing these types of an-
tibodies. To add to this complexity, when using surrogate approaches, both the biology and
binding affinities for each of the target antigens (tumor antigen and CD3) need to be similar
to the clinical candidate in order to appropriately safety in pharmacologically active nonclin-
ical species need to be developed in order to better predict potential safety liabilities with
these molecules.
Case Example 2. Blinatumomab (CD19/CD3 TDB, BiTE format):
Blinatumomab (MT103) is another example of a multitargeting T-cell engager molecule that
was designed to direct cytotoxic T cells to CD19-expressing cancer cells (Bargou et al., 2008).
CD19 is expressed on the surface of most human B-cell malignancies at high frequency
T celledependent bispecific antibody biotherapeutics 39
I. Development of biopharmaceuticals
(Scheuermann and Racila, 1995). Characteristics of MT103 include high affinity for CD19 and
low affinity for CD3. It is unclear whether high or low affinity for CD3 has a direct correlation
with T-cell activation. However, in this example, no T-cell activation by MT103 was observed in
the absence of B cells even at very high concentrations of the TDB (Schlereth et al., 2006). Since
MT103 was only cross-reactive to human and chimpanzee CD19 and CD3, the nonclinical
safety evaluation was conducted in chimpanzee at very low IV doses. Because of the mecha-
nistic concern for cytokine release via activation of T cells, this endpoint was evaluated both
in vitro and in vivo using human and chimpanzee PBMCs. The in vivo toxicology program
was conducted in two phases (Schlereth et al., 2006), the first being a dose escalation study
to identify a safe starting dose for the subsequent multiple dose study. For the dose escalation
study, a single female chimpanzee received escalating IV doses ranging from 0.06 to 0.12 mg/kg
MT103. In the multiple-dose study, a single female was used as a control, and one male and one
female were dosed at 0.1 mg MT103 on a weekly basis for a total of five infusions. Because of the
short half-life of MT103, a 2-h infusion was administered to achieve appropriate serum concen-
trations. Based on target antigen expression of CD19 on B cells, specific pharmacodynamic end-
points were included as part of the safety assessment evaluation. In order to determine the
effects of MT103 on peripheral B-cell counts, flow cytometric analysis was incorporated over
the course of the study (Schlereth et al., 2006). It should be noted that chimpanzees are no
longer considered viable species for toxicology studies (Van Norman, 2019).
The results of the safety evaluations demonstrated comparable in vitro cytokine release
(increases in IFNg, IL-2, and TNFa) in B-lymphoma cell line preparations in the presence
of MT103 and effector cells (PBMCs containing CD3þ T cells) from chimpanzee and human
(Schlereth et al., 2006). Cytokine release results from the in vivo experiments were consistent
with in vitro results. There was a marked, but transient increase in serum levels of IL-6, IL-2,
and IFNg 2e3 h after the start of the infusion. The highest serum cytokine values were
reached after the first infusion as compared to subsequent doses, which resulted in lower
serum cytokine levels. The expected PD effect of B-cell depletion was evaluated at select
time-points during the course of the study. Peripheral B-cell counts did not decrease during
the treatment phase, however, during the posttreatment phase, MT103 induced up to
40%e70% B-cell depletion (as compared to prestudy values) 3e5 weeks after the last infusion
(Schlereth et al., 2006).
From the repeat-dose study, cytokine release was consistent with the dose escalation study
where levels of cytokines decreased with subsequent dose administration. The initial increase
of cytokines followed by decreasing cytokine levels with repeated administration of MT103
was consistent with a first-dose effect. Some of the clinical indicators of cytokine release
also manifested acutely in the in vivo chimpanzee study as elevated body temperature and
initial cytokine release between in vitro and in vivo studies and the observation of a first-
dose effect that could be dampened with subsequent doses in single- and repeat-dose
in vivo studies. As observed after a single dose, maximal B-cell depletion in the periphery
was observed 2 weeks after the last infusion was administered. Based on the in vivo safety
profile in chimpanzee, these points were taken into consideration for management scenarios
in the clinical setting.
Blinatumomab was first explored in advanced refractory B-cell non-Hodgkin’s lym-
phomas (NHLs) (Bargou et al., 2008). Consistent with the short half-life of MT103, the clinical
program investigated escalating doses of 0.5e90 mg/m
2
per day administered by continuous
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals40
I. Development of biopharmaceuticals
infusion via a portable pump over 4e8 weeks (Nagorsen et al., 2011). The dose escalation in
the clinical studies was conducted beyond the doses in the in vivo toxicology studies. There-
fore, in comparison, doses used in the in vivo toxicology studies were several fold lower than
the Phase I clinical MTD. Some common adverse effects of blinatumomab in NHL/ALL pa-
tients include infections, pyrexia, headache, infusion reactions, and hematological toxicity
(Bargou et al., 2008; Jain & Litzow, 2020; FDA package insert Blincyto). Serious adverse
events include CRS and neurological related toxicities, which are managed by corticosteroid
prophylaxis, step up dosing regimens and on-treatment corticosteroids, treatment discontin-
uation, or cytokine blockers such as tocilizumab (Jain & Litzow, 2020). Most CNS events (im-
mune effector cell-associated neurotoxicity syndrome [ICANS]) occurred within the first
cycles of treatment and are a common cause for treatment interruption/discontinuation.
Although CNS toxicity was not reported with MT103 in chimpanzee studies, a few consid-
erations should be taken into account. The inability of preclinical toxicology models to accu-
rately predict certain clinical toxicities (e.g., CNS) as well as the limitations of using
chimpanzee as a toxicology species may have affected the ability of the toxicology program
to predict CNS adverse events. More robust toxicology evaluations could have been conduct-
ed with the availability of cross-reactive antibodies to orthologs in additional toxicology spe-
cies further enabling a more robust nonclinical toxicity evaluation. Consistent with the
catumaxomab program, use of steroids allowed fo r continued dose escalation when certain
adverse events occurred in Phase I. As with most development programs, the administration
of appropriate treatment regimens could mitigate certain adverse events to allow for
advancement of the clinical program.
Case Example 3. Cetuximab-based EGFR/CD3 TDB (BiTE format):
Epidermal growth factor receptor (EGFR) type 1 is a target for colorectal cancer. EGFR-
blocking monoclonal antibodies, such as cetuximab (Erbitux) and panitumumab (Vectibix)
are marketed for the treatment of patients with colorectal cancer (Rivera et al., 2008). Cetuxi-
mab, a human/mouse chimeric IgG1 antibody, has been shown to lyse EGFR-expressing
lung cancer cells by ADCC (Kurai et al., 2007). An EGFR-specific BiTE antibody was generated
based on the variable domains of cetuximab and panitumumab for the treatment of colorectal
cancer (Lutterbuese et al., 2010).
This final TDB case example illustrates the evolving development of this class of mole-
cules, allowing for a more robust preclinical safety evaluation of the clini cal candidate bio-
pharmaceutical. In this example, the biopharmaceutical not only binds in human but also
binds to cynomolgus monkey EGFR and CD3 target antigens thus allowing a more appro-
priate nonclinical toxicology evaluation (Lutterbuese et al., 2010). Cetuximab- and
panitumumab-based BiTE antibodies (C-BiTE and P-BiTE) had similar binding affinity and
activity to both cynomolgus monkey and human EGFR and CD3, thereby making interpre-
tation of the toxicology program relevant in predicting potential clinical outcomes.
The C-BiTE antibody was administered to cynomolgus monkeys by continuous IV infusion
for 3 weeks. The justification of a 3-week treatment duration was based on the expected poten-
tial for cetuximab to induce skin toxicity during this duration of treatment (Lutterbuese et al.,
2010). Serum C-BiTE levels at 12.4 mg/kg per day were similar to effective concentrations
from in vitro cytotoxicity assays. The results from the study indicate that C-BiTE was well toler-
ated at doses of 6.2 or 12.4 mg/kg per day for the entire 3-week period (Lutterbuese et al., 2010).
T celledependent bispecific antibody biotherapeutics 41
I. Development of biopharmaceuticals
Adverse clinical observations included a transient increase in body temperature, leukopenia,
and increased hepatobiliary parameters (Lutterbuese et al., 2010). At doses 31 mg/kg per
day severe signs of toxicity were observed within 56 h after the start of infusion, which led
to termination of animals for welfare reasons. Histopathological analysis of animals receiving
C-BiTE at 31 mg/kg per day demonstrated liver and kidney toxicity, possibly due to the
mechanism of action of C-BiTE and expression of EGFR in these tissues. Consistent with other
TDBs, animals dosed with 31 mg/kg per day showed increased levels of inflammatory cyto-
kines in serum (i.e., TNFa, IFNg, IL-6, IL-5, and IL-2). Similar to EpCAM and CD19-targeting
PVBs, the primary liabilities identified from the nonclinical studies for C-BiTE were consistent
with the effects of cytokine release. However, the ability to make comparisons of safety signals
from the preclinical studies in cynomolgus monkey to the clinical program is not possible at
this time as clinical data for C-BiTE is currently unavailable.
Case Example 4: Formation of ADAs limited the conduct of sub-chronic
(3-month) or chronic toxicology studies
Teclistamab (BCMA/CD3)
Teclistamab is approved for relapsed or refractory multiple myeloma and is an IgG4-based
bispecific antibody, targeting B-cell maturation antigen (BCMA). BCMA is relatively
restricted to cells of the B-cell lineage in normal tissues. A 5-week repeat dose study was con-
ducted in cynomolgus monkeys at dose levels of 1e30 mg/kg (FDA, 2023a). NHP was
considered the appropriate species based on cross-reactivity of the target antigens despite
the 36 affinity difference of NHP BCMA to human. No pharmacodynamic activity or
adverse effects were seen at dose ranges expected to be pharmacologically relevant. This
was attributed to the affinity difference and potential impact of ADA on exposures. ADA for-
mation was seen in 21/30 animals dosed with a significant impact on exposure. A 13-week
study was not conducted due to the lack of activity in NHP and the induction of neutralizing
ADAs in the 5-week study.
Epcoritamab (CD20/CD3)
Epicoritamab is an IgG1 with abrogated Fc effector function, targeting CD20 on B-cells
and CD3 on the T-cells. This CD20CD3 bispecific antibody is indicated for relapsed or re-
fractory diffuse large B-cell lymphoma (DLBCL) with clinical adverse events including
CRS, ICANS, infections, and cytopenias (FDA, 2023b). The 5-week toxicology assessment
in NHP included weekly i.v injections at dose ranges of 0.01, 0.1, 1 mg/kg, single i.v infu-
sion at 0.1 and 1 mg/kg, and monthly s.c injections at doses of 0.1e10 mg/kg. One animal
in the i.v high-dose group was euthanized due to cytokine- related adverse reactions. Over-
all, the nonclinical findings were consistent with the pharmacological activity and included
reversible hematological changes, B -cell depletion, and decreased lymphoid cellularity in
lymphoid tissues. Subcutaneous administration was shown to cause lower cytokine levels
relative to i.v administration. ADA formation was prevalent in both i.v- and s.c-dosed an-
imals with subsequent loss of exposure. All animals in the low-dose groups that were
dosed weekly formed ADA and some in the high 1 mg/kg dose group also showed
ADA formation with loss of exposure postday 15. Due to the extensive ADA formation
and subsequent loss of exposure in the 5 week repeat dose nonclinical study a 3-month
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals42
I. Development of biopharmaceuticals
repeat dose study was not deemed scientifically meaningful or warranted to support a BLA
submission.
As new targets are identified with cross-species target antigen binding and current mole-
cules enter and progress in the clinical setting, a further understanding of the translatability of
findings from nonclinical toxicology studies to the clinic will be determined for this class of
biopharmaceuticals.
Based on the case examples reviewed, the observation of cytokine release and its subse-
quent sequelae are safety liabilities associated with TDBs. This is not unexpected based on
the mechanism of action and resultant T-cell activation. Nonclinically, these acute toxicities
can be dose limiting and adaptive study designs such as longer infusions, step up dosing reg-
imens, or s.c administration could mitigate these toxicities similar to what is employed clin-
ically (Staflin et al., 2020; FDA, 2023a,b; Van De Vyver et al., 2021). When toxicities were
observed in the clinical programs, therapeutic intervention with steroids and anticytokine
treatments can be utilized to mitigate certain clinical adverse events to continue the study
as well as step up dosing regimens (Van De Vyer et al., 2021; Choudhry et al., 2018).
As demonstrated in these case examples, specific pharmacodynamic (PD) endpoints based
on the target tumor antigen should be incorporated as part of nonclinical toxicity evaluations.
For example, MT103 included PD assessment via immunophenotyping (flow cytometry) as
part of the nonclinical toxicology evaluation. Further, the PK, immunogenicity, desired clin-
ical route, and dosing schedule (e.g., step up dosing, s.c dosing) should also be considered
when designing nonclinical toxicology studies. Importantly, the immunogenici ty in nonclin-
ical species may limit the conduct of longer-term studies. In addition, the availability of an-
tibodies with antigen binding to both target antigens in nonclinical species and humans at
similar affinity increases the utility and relevancy of nonclinical toxicology studies to predict
potential clinical safety liabilities.
Dual-targeting antibodies
As was discussed in the previous sections, there are multiple platforms that are currently
being developed for PVBs. In this section, case examples reviewed will include bio-
pharmaceuticals that take advantage of targeting multiple parallel pathways addressing
oncology, ocular and neurodegenerative diseases. Some of the examples include utilization
of a transport system that is distinct from the target pathway to gain entry to an otherwise
difficult-to-access area of the body in a format that is reminiscent of full-length human IgG
with minimal structural or sequence deviations.
Oncology
Case Example 5. Anti-ErbB2/ErbB3 dual-targeting antibody:
Anti-ErbB2/ErbB3 (Merrimack/Amgen, MM-111) is an example of a dual-targeting anti-
body that takes advantage of the clinically relevant and validated target involvement of
the ErbB2 (also known as HER2) pathway in breast and other cancers (Tan and Yu, 2007; San-
tin et al., 2008). ErbB3 (also known as HER3), mentioned in the previous section, triggers
PI3K/AKT signaling upon ligand binding and heterodimerization with ErbB2 (Graus-Porta
et al., 1997). The idea of combining these two targets arose from the development of
Dual-targeting antibodies 43
I. Development of biopharmaceuticals
resistance against ErbB2-targeted therapy, such as trastuzumab (Nahta, 2006) and the subse-
quent upregulation of ErbB3 in the se resistant tumors (Sergina et al., 2007; Huang et al., 2010).
It is generally thought that a potentiation of efficacy is obtained when two receptors within
the same growth factor receptor pathway are blocked compared to a singly targeted receptor.
Using a Network Biology approach (Schoeberl et al., 2010), computational simulation of
ErbB2 receptor and subsequent PI3K-activated signaling predicted that simultaneous inhibi-
tion of receptor activation by ErbB3 would result in more optimal pathway inhibition and
also be more effective in ErbB2-amplified tumors. Therefore, a bispecific antibody consisting
of a ErbB2 docking arm and an ErbB3 effector arm that is linked via a modified human serum
albumin sequence was generated to block ligand-induced ErbB3 oncogenic signaling in
ErbB2-overexpressing tumor cells (McDonagh et al., 2012).
The potency of MM-111 was initially tested on a panel of tumor cell lines expressing vary-
ing amounts of ErbB2. While the IC50 values for inhibition of ErbB3 signaling were in the low
nanomolar range, MM-111 was able to more strongly inhibit ligand-activated phosphory-
lated ErbB3 to basal levels when ErbB2 expression levels were high.
When compared to humans, the extracellular domains of ErbB2 and ErbB3 of the rat and
monkey were determined to be 85%e99% homologous. In addition, binding affinities of MM-
111 to rat, monkey, and human ErbB2 and ErbB3 were relatively similar. Thus, target recep-
tor homology and pharmacological relevance had defined the rat and cynomolgus monkeys
as appropriate and selective species for toxicity testing.
To characterize the toxicities associated with MM-111, an initial preclinical 2-week repeat-
dose study was conducted in cynomolgus monkeys at doses up to 200 mg/kg (five doses
over 2 weeks, fo llowed by a 2-week recovery period). Ther e were no significant findings
that were identified in most animals with the exception of possible periarterial inflammation
in one recovery group female. Subsequently, 4-week repeat-dose studies in cynomolgus mon-
keys (once-weekly dose 5; up to 250 mg/kg; 4-week recovery) and in rats (once-week ly
dose 5; up to 500 mg/kg; 4-week recovery) were also conducted. Mild liver findings (peri-
ductal mixed cell infiltrate) were observed in the rats with no measurable effect on liver func-
tion. In the monkeys, with the exception of a single mortality in the high-dose males after the
fourth dose, minimal to moderate alterations in several parameters were noted. Namely,
there were decreases in body weight and similar to the rodents, mixed cell infiltrates were
observed in various tissues without significant impact on measurable organ function param-
eters. The no-observable-adverse-effect level was established to be 30 mg/kg based on the
arteritis in various tissues observed in monkeys. As a resu lt of these preclinical toxicity
studies, the clinical development plan of MM-111 entailed once-weekly dosing (2- or 4-
week cycle) with a starting dose of 3 mg/kg. This molecule was subsequently discontinued
due to lack of efficacy in phase I clinical trials for advanced refractory HER2þ breast cancer
(Olivier, 2012).
Case Example 6. Anti-HER3/EGFR dual-targeting antibody:
Anti-HER3/EGFR (Genentech, MEHD7945A) is an example of a high-affinity dual-action
antibody that inhibits cancer cell growth stimulated by signaling of both EGFR and HER3
pathways. Activation of EGFR leads to homo- or hetero-dimerization and subsequent activa-
tion of intracellular tyrosine kinases that regulate, among other things, cell cycle progression.
HER3 is of the dimerization partners of EGFR and can potently initiate signaling along the
PI3K/AKT cell survival pathway. Dysregulation of HER family signaling, including signaling
through EGFR and HER3, has been implicated in processes related to cancer (Hynes and
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals44
I. Development of biopharmaceuticals
MacDonald, 2009). The proposed mechanism of action of the dual-action anti-HER3/EGFR
antibody is inhibition of ligand binding to the receptors and a restriction on the extracellular
domain conformation, resultin g in the inhibition of ligand-dependent HER signaling (Schaefer
et al., 2011). This approach could be effective in multiple cancer types and may limit emergence
of drug resistance. While some studies have suggested that one potential mechanism of ac-
quired resistance to EGFR-targeted therapies is the dysregulation of HER family ligands or
other HER family receptors (Kong et al., 2008; Yonesaka et al., 2011), HER3 activation has
also been implicated in resistance to cetuximab (anti-EGFR monospecific therapeutic antibody)
and HER-targeted tyrosine kinase inhibitors in preclinical studies (Schoeberl et al., 2010).
The novelty of the MEHD7945A molecule arises from the presence of two identical Fab
arms that can each bind either of two target antigens, EGFR and HER3, with high affinity,
simultaneously blocking ligand binding to EGFR and HER3. Resul ts of nonclinical binding
specificity assessment conclude d that MEHD7945A had a higher binding affinity for HER3
than for EGFR (Schaefer et al., 2011).
As part of the nonclinical development program, pharmacology and efficacy studies were
conducted. Results of these studies con firmed that dual blockade of EGFR and HER3 was
equally or more efficacious than monospecific anti-EGFR (cetuximab) in multiple tumor
growth models.
To determine safety for this molecule preclinically, cynomolgus monkeys were chosen as
the relevant species as MEHD7945A was cross-reactive to both monkey HER3 and EGFR. In
addition, due to the binding specificity of MEHD7945A to mouse EGFR, but not to mouse or
rat HER3, rodents were not considered appropriate species for toxicity testing. Because of the
role of EGFR in epidermal differentiation, antagonism of EGFR signaling is known to cause
dermatologic toxicity. Preclinical studies in monkeys were predictive of dermatologic toxic-
ities from EGFR inhibition that has been recapitulated in the clinic in patients administered
cetuximab (Wong, 2005). Therefore, the main concern of dual blockade of HER3 and EGFR
was the potential for unique safety liabilit ies and/or exacerbation of known toxicities
(EGFR-mediated dermatological and gastrointestinal toxicity). These concerns were
addressed in the toxicology studies in cyno molgus monkeys. When MEHD7945A was tested
alongside cetuximab at comparable doses to determine the relative extent of toxicity and tox-
icokinetics of this molecule in monkeys, MEHD7945A elicited less dermatologic toxicity than
cetuximab. At equivalent doses, few er monkeys treated with MEHD7945A developed rash
and moist dermatitis at a later time and with less severity than monkeys treated with cetux-
imab alone (Schaefer et al., 2011).
Coagulation disorder
Hemophilia
Case Example 7. Emicizumabdactivated human coagulation factor IX (hFIXa) factor X
(hFX):
Emicizumab was developed for routine prophylaxis to prevent or reduce the frequency of
bleeding episodes in adult and pediatric patients with hemophilia A. Patients with hemophil-
ia A lack factor VIII, a substance in the body that helps the blood to clot. Emicizumab is a
Dual-targeting antibodies 45
I. Development of biopharmaceuticals
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