Critical Reviews in Oncology/Hematology 92 (2014) 153165

Bispecific antibody platforms for cancer immunotherapy

Roeland Lameris a , Rene C.G. de Bruin a , Famke L. Schneiders a ,
Paul M.P. van Bergen en Henegouwen b , Henk M.W. Verheul a ,
Tanja D. de Gruijl a , Hans J. van der Vliet a,
b

a Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Accepted 8 August 2014

Contents
1.
2.

3.

4.
5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Currently available bispecific antibody platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Trifunctional hybrid antibodies (Triomab) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1. Catumaxomab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2. Ertumaxomab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3. FBTA05 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Single chain variable fragment (scFv) based platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Tandem scFv (TaFv). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Bispecific diabodies (bsDb) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Other bispecific antibody based platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Limitations of the type of bsAb constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Immunogenicity of bispecific antibody constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Size of the construct: Pay off between circulation half-life time and tumor penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Antibody construct stability and manufacturing difficulties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perspectives related to the bsAb construct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Targeting specific lymphocyte subsets to maximize efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Invariant natural killer T-cells (iNKT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Natural killer cells (NK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract
Over the past decades advances in bioengineering and expanded insight in tumor immunology have resulted in the emergence of novel
bispecific antibody (bsAb) constructs that are capable of redirecting immune effector cells to the tumor microenvironment. (Pre-) clinical
studies of various bsAb constructs have shown impressive results in terms of immune effector cell retargeting, target dependent activation

Abbreviations: VH , variable heavy chain domain; VL , variable light chain domain; mAb, monoclonal antibody; bsAb, bispecific antibody; scFv, single
chain variable fragment; Triomab, trifunctional hybrid antibody; TaFv, tandem single chain variable fragment; BiTE, bispecific T-cell engager; bsDb, bispecific
diabody; scDb, single chain diabody; DART, dual affinity retargeting molecule; VHH, variable domain of heavy chain-only Ab.
Corresponding author. Tel.: +31 20 4441295; fax: +31 20 4444355.
E-mail address: jj.vandervliet@vumc.nl (H.J. van der Vliet).
http://dx.doi.org/10.1016/j.critrevonc.2014.08.003
1040-8428/ 2014 Elsevier Ireland Ltd. All rights reserved.

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R. Lameris et al. / Critical Reviews in Oncology/Hematology 92 (2014) 153165

and the induction of anti-tumor responses. This review summarizes recent advances in the field of bsAb-therapy and limitations that were
encountered. Furthermore, we will discuss potential future developments that can be expected to take the bsAb approach successfully forward.
2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bi-specific antibodies; Dual specific retargeting; Immune effector cells; Anti-cancer therapy

1. Introduction

2. Currently available bispecic antibody platforms

Several clinically available therapeutic monoclonal antibodies (mAbs) can induce immune-mediated tumor cell
killing through mechanisms that include complementdependent cytotoxicity (CDC) and antibody-dependent
cellular cytotoxicity (ADCC). Following binding of the mAb
to its tumor target, interactions of the Fc-portion with Fcreceptors (FcR) expressed by effector cells (e.g. natural
killer (NK) cells, macrophages and T-cells) may result
in CDC and ADCC and subsequent antitumor cytotoxicity and/or phagocytosis. In clinical series, ADCC has been
demonstrated to significantly enhance the efficacy of various mAbs, including rituximab (anti-CD20), trastuzumab
(anti-human-epidermal-growth-factor receptor 2 (Her2)) and
cetuximab (anti-epidermal-growth-factor receptor (EGFR))
[1]. Although data are inconsistent, clinical responses may
be influenced by FcR polymorphisms [2]. Granting their
therapeutic efficacy can in part be attributed to beneficial
secondary immune effects, it is clear that mAbs still do not
exploit the full potential of the immune system as effects are
e.g. hampered by circulating immunoglobulins (Ig) competing for FcR binding spots on immune effector cells, and
inadequate tumor-target penetration due to their relatively
large size (150 kDa) [3]. Furthermore, binding to inhibitory
FcR on immune cells may result in internalization of the
mAb-tumor target-FcR complex reducing its therapeutic
efficacy [4].
Bispecific Abs (bsAbs), capable of binding two targets
simultaneously, lack several of the above described limitations and can potentially induce a more powerful anti-tumor
immune response. The first bsAbs were engineered by either
chemical crosslinking or exchange of different heavy chains
as a result of fusion of two hybridoma cell lines (i.e. hybrid
hybridomas or quadromas). Despite some clinical effects,
none of these bsAbs made it to advanced stage clinical
trials, as a low production yield owing to random association of heavy/light chains and immunogenicity caused by
human anti-mouse/rat antibodies (HAMA/HARA) severely
hampered clinical applicability [5]. It was not until 1995
that bsAb research was sparked again by the introduction of
fused mouse-rat hybrid antibodies and tandem single-chain
variable fragments (TaFv) [6,7].
Here, we will review the main bsAb formats that are
currently being developed for tumor retargeting of immune
cells and discuss thus far obtained (pre-)clinical results and
encountered limitations. Furthermore, we will elaborate on
potential ways to take the bsAb approach forward.

2.1. Trifunctional hybrid antibodies (Triomab)

Introduced in 1995, this platform offered a solution to the
random association of heavy/light chains observed in classic quadroma technology. By combining the halves of two
distinct antibodies, a tumor-specific mouse IgG2a and a CD3specific rat IgG2b, a full-size functional mAb was engineered
and termed Triomab (Trion pharma Inc.) (Fig. 1b). Due to
species-preferential heavy/light chain pairing random association was greatly reduced. Interestingly, the hybrid mouse/rat
Fc-portion was able to activate FcR+ accessory cells [6,8,9].
Preclinical studies with a Triomab targeting epithelial cell
adhesion molecule (EpCAM), expressed by the majority of
epithelial cancers, and CD3 expressed by T-cells, demonstrated redirection and activation of T- and accessory cells
(e.g. NK cells, dendritic cells (DC) and macrophages). T-cell
activation was complemented by the induction of T-cell mediated tumor lysis, cytotoxic cytokine release, and ADCC in
the picomolar range [8,9]. The additive value of a functional
Fc-portion was underscored by enhanced tumor protection
in mice treated with a Triomab compared to a similar bsAb
consisting of two chemically cross-linked fragment antigen
binding (Fab) regions (F(ab)2 ) (Fig. 1c). In vivo assessment
of the Triomab injected intraperitoneally (i.p.) in a syngeneic
C57BL/6 and BALB/c mouse model using (human) EpCAM
expressing B16-melanoma and A20-lymphoma cells, respectively, demonstrated a significantly improved tumor cell
elimination compared with the simultaneous administration
of both parental antibodies. In the Triomab treated group
a 100% survival rate, complete tumor eradication and protection against tumor rechallenge was reported. Selective
depletion of both CD4+ and CD8+ T-cells resulted in a marked
loss of tumor protection and survival. Of note, only the animals injected with human EpCAM expressing tumors and
Triomab treatment developed strong anti-EpCAM specific
humoral immune responses [10], with additional evidence
suggestive of epitope spreading. These data resulted in the
development and clinical evaluation of a number of Triomabs,
including catumaxomab, ertumaxomab and FBTA05 which
will be discussed below.
2.1.1. Catumaxomab
Catumaxomab, an anti-EpCAM-anti-CD3 Triomab, was
the first Triomab studied in patients. A phase-I trial of a single intravenous (i.v.) dose of catumaxomab in patients with
non-small cell lung cancer (NSCLC), established a maximum

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155

vascular endothelial growth factor (VEGF), increased levels

of activated CD4+ and CD8+ T-cells as well as an elimination
of CD133+ /EpCAM+ cancer stem cells (CSC) compared to
both baseline levels and non-treated control patients [14].
Importantly, compared to control patients catumaxomab
resulted in a significant prolongation of puncture-free survival (median 11 vs 46 days, p < 0.0001) and an increased
time to next paracentesis (median 13 vs 77 days), precluding approximately 5 therapeutic paracentesis procedures.
Likewise, symptoms and signs related to ascites were significantly reduced. Although a positive trend was observed in
improvement of median overall survival (72 (95% CI: 6198)
versus 68 days (95% CI: 4981), this only reached statistical significance in patients with gastric carcinoma (median
71 vs 44 days; p = 0.0313) [15]. Notably, HAMA/HARA
development, which strongly correlated with clinical endpoints, was observed in the majority of patients (>70%)
after the last treatment cycle and will be discussed in more
detail in Section 3.1 [16]. Adverse effects (AE) were most
commonly cytokine release-related (i.e. pyrexia, nausea and
vomiting), manageable and generally reversible. Reported
Common Toxicity Criteria (CTC) grade 3 AE considered to be treatment related included abdominal pain (9.6%)
and lymphopenia (7.0%). Reported serious AE included
ileus (3.2%) and gastric hemorrhage (0.6%) [15]. Based
on these data, catumaxomab was approved by the European Medicine Agency (EMA) for the i.p. treatment of
malignant ascites in patients with EpCAM-positive carcinomas where standard therapy is not available or no longer
feasible.

Fig. 1. Variable heavy chain domains (VH ) are depicted in dark blue and
dark orange, variable light chain domains (VL ) are depicted in light blue
and light orange. Orange and blue indicate arms with different specificities.
Peptide linkers are shown as gray lines. (a) mAb, monoclonal antibody; (b)
Triomab, bispecific rat/mouse antibody; (c) F(ab)2 , two chemically crosslinked fragment antigen binding (Fab) regions; (d) scFv, single chain variable
fragment; (e) TaFv, tandem single chain variable fragment; (f) bsDb, bispecific diabody; (g) scDb, single chain diabody; (h) DART, dual affinity
retargeting molecule; (i) Heavy chain-only antibody; (j) bsVHH, bispecific
variable domains of heavy chain-only Ab. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)

tolerated dose (5 g with 40 mg dexamethasone) and reported

favorable survival in several patients with advanced-stage disease surviving past 26 months. None of the patients developed
HAMA or HARA within 28 days after infusion [11].
More extensive studies have evaluated i.p. administration of catumaxomab in patients with malignant ascites
due to various EpCAM positive tumors. In a randomized
phase II/III study, involving heavily pre-treated patients with
symptomatic malignant ascites, infusions were given with
an increasing dose (10150 g) on days 0, 3, 7 and 10
[12,13]. Analyses of ascites demonstrated decreased levels of

2.1.2. Ertumaxomab
Ertumaxomab, an anti-Her2-anti-CD3 Triomab, was
demonstrated to initiate effective immune mediated cytotoxicity against Her2 expressing tumor cell lines in vitro
and to efficiently lyse low Her2 expressing target cells
where trastuzumab, a mAb targeting Her2, was completely
ineffective [17]. In a phase I trial, i.v. administration
of 3 ascending doses (10200 g) of ertumaxomab on
days 1, 7 and 13 to patients with metastatic breast cancer induced strong inflammatory reactions with clinical
responses in some patients. The severity and number of
AEs were dose related, with dose limiting events being
immune related. Reported serious AE classified to be treatment related included fully reversible severe hypotension
(5.9%), systemic inflammatory response syndrome (5.9%)
and, aggravation of congestive heart failure (5.9%). CTC
grade 3 toxicities included fully reversible lymphocytopenia (76%) and elevation of liver enzymes (47%). Of
note, the number of CTC grade 3 toxicities and serious
AE seemed to be dose related. Within 6 weeks after the
first dose, HAMA/HARA were observed in 25 and 31%
of patients, respectively [18]. A phase II trial was terminated prematurely due to changes in company development
plans.

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2.1.3. FBTA05
FBTA05, an anti-CD20-anti-CD3 Triomab, demonstrated
effective CD20+ lymphoma killing in vitro. A pilot study
involving patients with recurrent/refractory non-Hodgkin
lymphoma (NHL) or chronic lymphatic leukemia (CLL)
treated with i.v. FBTA05 followed by donor lymphocyte infusion or peripheral blood stem cell transplantation showed a
prompt but transient response in several patients [19]. A phase
I/II study is ongoing (NCT01138579).
2.2. Single chain variable fragment (scFv) based
platforms
Some of the mentioned limitations of whole antibodies can
be overcome by reducing antibodies to their minimal binding
domains. Advances in genetic engineering have provided a
vast amount of such antibody like constructs, including small
antibody fragments. One such fragment, termed single-chain
Fv (scFv), is made by the association of the variable parts
of heavy (VH ) and light (VL ) chains through a peptide linker
(Fig. 1d) [20]. Two formats based on scFvs have been studied extensively, namely tandem scFv (TaFv) and bispecific
diabodies (bsDb).
2.2.1. Tandem scFv (TaFv)
By covalent bonding of two scFvs with a flexible peptide linker in a tandem orientation, a bispecific TaFv can be
formed (Fig. 1e). It is expected that the flexible orientation
between the two binding domains enhances simultaneous target ligation, while their small size (5560 kDa) allows rapid
tumor penetration [3].
One type of TaFv, made by fusing an anti-CD3 to an antitumor associated antigen (TAA) scFv via a 5 residue peptide
linker (GGGGS), has been extensively studied. These TaFv,
termed bispecific T-cell engagers (BiTE) (Micromet Inc.) can
effectively redirect and activate polyclonal T-cells in vitro
resulting in a highly cytotoxic response at pico- to femtomolar concentrations in the absence of co-stimulation [21,22].
Video microscopy revealed that BiTEs allowed serial killing
of target cells by engaged T-cells with complete elimination of target cells at effector-to-target ratios of 1:5, which is
24 times lower than ratios needed for Triomabs [23]. Target
cell lysis involved the formation of tight cytolytic synapses
between target- and effector cells and the subsequent release
of cytotoxic effector molecules (e.g. perforin and granzyme
B) [24]. As T-cell activation depended on multivalent binding of BITEs due to their low affinity for CD3, activation
occurred in a strictly target dependent fashion [25].
2.2.1.1. Blinatumomab. Blinatumomab, being the first and
most advanced BiTE in clinical studies, is derived from
murine scFvs targeting human CD19 and CD3. Several
clinical studies evaluated its efficacy in various types of Bcell NHL and leukemia. Complete (CR) and partial tumor
responses (PR) were demonstrated upwards of a dose of
15 g/m2 /24 h, given as continuous infusion over 4 or 8

weeks. Importantly, continuous infusion was required to

ensure sustained effective serum levels due to a very short
serum half-life time of less than 2 h due to renal excretion
[26,27].
Based on results in B-NHL, a phase II study was initiated
in patients with persistent/relapsed minimal residual disease
(MRD) positive B-cell acute lymphatic leukemia (B-ALL).
Patients were treated with a continuous 4-week i.v. infusion at
a dose of 15 g/m2 /24 h every 6 weeks with responders being
permitted to receive up to 3 consolidation cycles. Sixteen out
of 20 patients became MRD-negative at the end of the first
cycle, 12 of which had molecular refractory disease to previous treatment [28]. Long term follow-up (median 33 months)
demonstrated a hematologic relapse-free survival in 12 out
of 20 patients [29]. In all 20 patients T-cells declined rapidly
but recovered within days to above baseline levels, with a significant percentage of reappearing CD8+ and CD4+ T-cells
transiently expressing the early activation marker CD69. Bcell counts dropped below 1 cell/L and did not recover until
after therapy cessation. The decline in B-cell counts most
likely resulted from redirected cell lysis [30]. Similar results
were reported in an interim analysis of a phase II trial in
relapsed/refractory B-ALL. In this study the starting dose
was reduced (5 g/m2 /24 h in the first week) to prevent AE.
Seventeen out of 25 patients achieved CR or CR with partial
hematological recovery [31].
Most common AEs with blinatumomab included flu-like
symptoms during the first days of treatment which resolved
under ongoing infusion. AEs correlated with peak levels of
activated CD8+ and CD4+ T-cells and with increased serum
cytokines levels. Commonly reported CTC grade 3 AE in
the different studies included lymphopenia (up to 77%) and
leukopenia (up to 47%). More serious AEs, although completely reversible, included neurological AE (e.g. seizures
and encephalopathy in up to 4.8% and 8.6% of patients,
respectively) presumably caused by released cytokines and
frequently resulted in treatment interruption or discontinuation. Also, cases of hemophagocytic lymphohistiocytosis
(HLH) presumably caused by blinatumomab treatment have
been described [2630,32]. Mitigating measures, including
lower starting doses, a double-step dose increase and glucocorticoid administration, were protective and allowed for
treatment continuation or resumption [2630].
Multiple trials designed to determine the optimal dose,
treatment schedule and clinical efficacy of blinatumomab
are ongoing (NCT01741792, NCT01471782, NCT01207388
and NCT01466179).
2.2.1.2. Alternative TaFv constructs. TaFv constructs targeting EpCAM and prostate specific membrane antigen
(PSMA) have entered phase I clinical trials in epithelial and prostate cancer, respectively (NCT00635596 and
NCT01723475). A multitude of other TaFvs is being evaluated pre-clinically, including constructs targeting EGFR [33],
carcinoembryonic antigen (CEA) and CD33. Of interest, an
anti-prostate specific cell antigen (PSCA)-anti-CD3 TaFv

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was recently successfully humanized without compromising

its in vitro and in vivo characteristics. Humanization may
prohibit HAMA associated AE [34].
Efforts have also focused on adding a third binding site to TaFv, thereby creating triple bodies (scTb).
Indeed, an anti-CD16-anti-CD33-anti-CD19 scTb demonstrated enhanced lysis of CD33-CD19-positive leukemic
cells in vitro, compared to both an anti-CD16-anti-CD19 and
an anti-CD16-CD33 TaFv [35].
2.2.2. Bispecic diabodies (bsDb)
In contrast to TaFvs, bsDbs are formed by non-covalent
association of two scFvs, consisting of a VH and VL domain
from different parent Abs, connected with a very short peptide
linker precluding intra-chain domain interactions (Fig. 1f).
bsDbs have their variable domains orientated in an opposed
fashion and are very rigid and small (60 kDa) [36]. Because
individual chains are not covalently associated, VH and VL
binding interactions remain a limiting factor in the stability of the molecule, possibly resulting in disintegration,
dimerization and aggregate formation. This can, at least
in part, be prevented by introducing an additional peptide
linker between the scFv, forming a single chain bsDb (scDb)
(Fig. 1g) [3739]. Alternatively, an increased stability can
be achieved by introducing a disulfide bond between the
C-terminus of each scFv, resulting in a Dual-Affinity ReTargeting (DART) molecule (Fig. 1h). DART molecules have an
extended storage and serum stability, do not form aggregates
and demonstrate potent in vitro and in vivo activity [40].
Notably, by varying the length of the additional peptide linker in scDbs, tetramers and tandem bsDbs can be
formed [37,4143] with higher affinity, a reduced target celldissociation rate and improved storage stability. Furthermore,
in vivo experiments showed higher serum retention compared
to bsDbs [41], possibly due to their increased affinity and size
(114 kDa) reducing renal clearance. Their increased size
may however decrease effective tumor penetration.
2.2.2.1. Pre-clinical studies. An anti-PSMA-anti-CD3
bsDb proved to be potent for CD3+ T-cell tumor targeting
specifically inducing lysis of PSMA-expressing prostate cancer cells in vitro and inhibiting human prostate cancer growth
in xenografted mice [42]. Similar results were reported for
an anti-CD19-anti-CD3 [44,45] and an anti-EGFR-anti-CD3
bsDb [46]. Direct comparison of an anti-EGFRanti-CD3
bsDb with its TaFv equivalent indicated an equal binding
affinity, but enhanced in vitro cytotoxicity in favor of the
TaFv [47]. As the compact structure of bsDbs might hinder
cross-linking, it is interesting to note that rearrangement
of the variable domains (e.g. varying the VH -linker-VL
order) can lead to superior in vivo results when compared
to the TaFv equivalent, though this in vivo superiority
may also result from the enhanced stability of bsDbs at
37 C [48].
The potency of DARTs was demonstrated in an in vitro
side-by-side comparison of blinatumomab and a DART

157

bearing identical CD3 and CD19 Fv-domains. The DART

molecule outperformed the BiTE molecule with respect
to both the induction of B-cell lysis and stimulation of
T-cell activation, with T-cell activation and proliferation
occurring solely in a target-cell dependent manner in both formats. Based on identical recognition specificities and similar
molecular masses, differences between the DART and BiTE
platform thus resulted from structural differences, e.g. in the
level of fixation between the two opposing binding sites. The
observed higher association rate and target affinity of DARTs
may have resulted in prolonged intercellular contacts creating a more robust cytotoxic T-cell response [49]. While bsDbs
yielded promising results in pre-clinical studies, with some
apparently outperforming TaFv, no clinical data have yet been
reported.
2.3. Other bispecic antibody based platforms
Although a number of alternative bispecific constructs
designed for tumor targeting of effector cells have been
engineered, to date only a few have been evaluated in preclinical studies [20,50]. These include, but are not limited
to, engineered human monospecific IgG1 and IgG2 subtypes
that allow, under appropriate redox conditions, heavy chain
exchange to form full-length bsAb [51], an anti-CD3 scFv
fused to a high-affinity monoclonal T-cell receptor (mTCR)
specific for a tumor-associated MHC-1 antigen (ImmTAC)
[52,53], a fusion protein consisting of a scFv targeting CEA
and the human UL16 binding protein 2 (ULBP2), which as an
NKG2D ligand can be used for targeting NKG2D-expressing
immune effector cells [54] and, a scFv fused to the N- and
C-terminus of a trimerizing scaffold domain, resulting in a
bispecific hexavalent trimerbody [55]. In, addition fusion
conjugates consisting of an anti-TAA coupled to an MHC
class I molecule containing a selected antigenic peptide have
been shown to be able to effectively redirect and activate
T-cells in vivo [56,57]. These platforms are not further discussed as limited preclinical efficacy data and no clinical
efficacy data are presently available.

3. Limitations of the type of bsAb constructs

3.1. Immunogenicity of bispecic antibody constructs
Historically, murine- and rat-derived mAbs have been
associated with short serum half-life time upon repeated
administration due to the formation of neutralizing
HAMA/HARA and the related susceptibility to trigger a
cytokine release syndrome (CRS) in patients. Indeed, as discussed, i.v. dosing of Triomabs was limited by immunological
AE and, HAMA/HARA formation may restrict repetition of
treatment cycles. Though humanization is generally thought
to limit anti-drug antibody (ADA) formation, it cannot
completely circumvent this, as was demonstrated by the
anti-TNF- mAb adalimumab, where up to 89% of patients

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R. Lameris et al. / Critical Reviews in Oncology/Hematology 92 (2014) 153165

developed neutralizing human anti-human Abs (HAHA). As

direct ligation of effector cells through e.g. FcR may also
trigger CRS, immune-related AEs of mAbs cannot always
be prevented [38,58,59]. Despite these drawbacks, it has
been shown for various mAbs that both ADA and CRS can
actually promote tumor destruction [16,60]. Indeed, HAMA
development in patients with malignant ascites treated with
catumaxomab correlated with prolonged puncture-free survival [16]. Although tentative, HAMA development may also
simply reflect a less suppressed immune system.
As indicated, targeting the FcR can enhance tumor
destruction through ADCC. Indeed, the combination
of an anti-CD19-anti-CD3 and an anti-CD19-anti-CD16
(FcRIIIA) bsDb had a synergistic effect [61]. Similarly an
amplified anti-tumor response was seen after adding a functional Fc-tail to a scDb [62]. However, as was observed in
in vitro studies with catumaxomab, simultaneous targeting
of FcR+ cells and T-cells may also result in cross-linkage
and activation of these cells in the absence of target antigen
[17,63] thereby limiting the intended efficacy of tumor targeting of these immune cells. Other tri-specific constructs that
cross-link effector cells may exhibit similar limitations.
By eliminating the Fc-region Abs are reduced in size
to their minimal binding domain (e.g. TaFvs and bsDbs)
and it is believed that they are therefore less immunogenic
[38]. In support of this, patients treated with (murine mAbderived) blinatumomab, developed no HAMA and had stable
steady-state levels over the course of treatment, indicating
low immunogenicity [27,28]. Of note, in this case lack of
ADA might also have been due to effective B-cell depletion. BsDbs may be even less immunogenic due to their very
compact size [37,42,45].
Concerns have been raised about ongoing T-cell activation during BiTE-treatment, with several patients developing
uncontrolled immune activation (i.e. CRS and HLH) [32].
Moreover, persistent T-cell activation may induce anergy,
though the observed continued B-cell suppression during treatment essentially reflects preserved T-cell function
[30].
3.2. Size of the construct: Pay off between circulation
half-life time and tumor penetration
The Fc-domain of Abs enhances the serum half-life time
(to >10 days) through neonatal-FcR mediated recycling by
e.g. endothelial cells; hence absence of the Fc-tail may
result in swift clearance from the circulation. Small sized
bsAb fragments (5060 kDa), such as TaFv and bsDb, also
allow for easy extravasation, renal filtration and degradation due to a glomerular filtration barrier (GBM) threshold
of 4050 kDa [3,38,64]. Indeed, continuous infusion of
BiTEs was required to maintain stable plasma levels and
maximize clinical efficacy [26]. At present no information
is available on the impact of such continuous infusions on
either treatment costs or patient quality of live, nevertheless prolonged exposure time is often considered critical

for optimal therapeutic efficacy [58]. Hence various methods have been successfully deployed to extend the serum
half-life time of small-sized constructs including PEGylation, N-glycosylation, fusion to human albumin (covalently
or using albumin-binding domains), or linkage to antiCD16 Ab fragments [64]. Linkage of an anti-CEA-anti-CD3
scDb to an albumin-binding domain resulted in a prolonged
half-life time and a 5-fold increase in accumulation in
xenografted CEA+ tumors in mice. Nevertheless, in this setting reduced cytotoxicity was observed [65], possibly due to
steric hindrance. Similar reductions in cytotoxicity have been
reported for other serum half-life time extending strategies
[64,65].
Compared to large IgG (150 kDa) molecules, small bsAb
fragments exhibit improved tumor penetration and display a
more homogeneous distribution within tumors; furthermore,
due to their multivalent nature bsAb fragments tend to have
high avidity with prolonged target retention [3]. Both superior tumor penetration and target retention may result in a
synergistic effect on tumor destruction.
Taking the above into consideration, especially solid and
bulky tumors may require strong and homogeneous tumor
penetration provided by small sized constructs. Due to their
enhanced tumor penetration, serum levels of small-sized
bsAb constructs might not necessarily reflect efficacy. Interestingly, rapid clearance from the circulation may reduce
non-specific (off-target) cytotoxicity due to favorable tumorto-blood ratios.
3.3. Antibody construct stability and manufacturing
difculties
As mentioned, species restricted heavy-light chain paring
in Triomabs resulted in a 3.5-fold higher production yield
compared to conventional quadromas and allowed for a single purification step. The in vivo stability of catumaxomab
appeared favorable, with up to 100% being immunologically
active after three days in ascites [66]. These properties greatly
facilitated production and clinical development [6]. Nevertheless, as with all whole mAbs, mammalian cells are often
needed as a host, increasing cost and production time [67].
scFv can be expressed in bacteria, but tandem (i.e. TaFv)
molecules tend to form insoluble aggregates in bacteria due
to incorrect folding and therefore require production in mammalian cells for high production yields [7,21,41]. bsDbs lack
this disadvantage, but since two different polypeptide chains
are expressed within one cell, inactive homodimers can be
produced alongside the active heterodimers. By introducing
an additional peptide linker (as in scDbs) or disulfide bond (as
in DARTs) homodimerization can be prevented. While scDb
can be expressed in bacteria, disulfide bonds reduce yields in
bacteria [37,38,40,68].
Though long-term stability of TaFvs may require additional engineering [40,69], scDb are 2-3-fold more stable than
TaFvs in human plasma at 37 C [70]. Biological properties
of scDb may however be strongly affected by even modest

R. Lameris et al. / Critical Reviews in Oncology/Hematology 92 (2014) 153165

variations in their composition (e.g. differences in linker

lengths or the order of variable domains) [38].

159

evaluated for immune effector cell retargeting, however based

on the above listed properties, bsVHHs may prove to be
very efficient. Future studies will have to determine if these
properties will translate to the clinic.

4. Perspectives related to the bsAb construct

As outlined, currently used bsAb constructs can induce
retargeting of immune effector cells and can result in therapeutic responses, yet improvements to overcome encountered
difficulties are required and appear feasible. Based on discussed limitations, several ideal characteristics of bsAbs can
be recognized: they should be (i) easy to manufacture at
relatively low cost, (ii) have a high serum and storage stability, (iii) be small (5060 kDa or less) to allow for rapid
and homogeneous tumor penetration, yet (iv) have a sufficiently long half-life time to induce a therapeutic effect,
(v) have high target-cell affinity to ensure tumor retention,
but (vi) safeguard strictly target-dependent activation and,
finally, (vii) be non-immunogenic. All previously discussed
bsAb constructs lack several of these characteristics to some
extent.
The variable domain of heavy chain-only Abs (Fig. 1i),
naturally occurring in the family of Camelidae, including llama, camel and dromedary [71,72], may fulfill many,
if not all, of these ideal characteristics. These variable
domains of heavy chain-only Abs (VHH) or Nanobodies
(Ablynx Inc.) (Fig. 1j) share the same large, diversified
and specific repertoire of Ag binding sites as conventional Abs but also allow specificity to unique epitopes,
including cryptic and not otherwise easily accessible epitopes, due to their distinctive three-dimensional structure
[3,73]. Specific VHHs are easily retrieved after panning of a
phage-displayed rearranged VHH -gene pool cloned from an
immunized camelid, and can inexpensively be produced in
bacteria, yeast or mammalian cells [74]. Solubility, aggregation and degradation problems often encountered with
scFvs, are prevented due to their single domain nature.
The relatively small size (15 kDa) permits easy linkage of
VHH into dimers (35 kDa), trimers (50 kDa) or multimers that retain rapid and homogeneous tumor penetration
[7578].
Owing to their size VHH are rapidly cleared from the
circulation. This can, however, be prevented by fusion to
a VHH targeting e.g. mouse/human albumin [79] or FcR.
Bio-distribution studies in mice with a radiolabeled bivalent
anti-EGFR-anti-albumin VHH demonstrated an extended
half-life time. Blood clearance was similar to that of
the anti-EGFR mAb cetuximab, whereas more deep and
homogeneous tumor penetration was observed using the antiEGFR-anti-albumin VHH (Ab-construct binding to 100% vs
60% of tumor cells) [79]. VHH amino acid sequences share
high homology with the human type 3 VH domain (VH 3),
most likely accounting for their low immunogenicity [72,78].
Still, VHH are usually humanized before clinical application [75] with preliminary clinical data thus far reporting no
ADA development [80]. As yet no bispecific VHH has been

5. Targeting specic lymphocyte subsets to maximize

efcacy
In order to effectively recruit and activate all T-cell subsets
and induce tumor cell lysis, most bsAbs target the uniform
CD3-portion of the TCR complex. Targeting T-cells is attractive due to their destructive potential, but carries a risk as was
strikingly demonstrated by TGN1412, a monospecific superagonistic CD28-mAb that induced a life threatening cytokine
storm in healthy volunteers [81]. Measures to preclude such
tragedies are required, and include strictly target dependent effector cell activation as a means of improving safety
[59].
Targeting of CD3 results in the recruitment of a wide
range of T-cells, including CD4+ , CD8+ , T-cells and
different immunoregulatory and immunosuppressive T-cell
subsets. T-helper 1 (Th 1-)cells are considered to be proinflammatory cells that play an important role in tumor immunity,
whereas Th 2-cells and most notably CD4+ CD25+ Foxp3+
regulatory T-cells (Tregs) may promote tumor growth. For
example, Tregs have been recognized for their deleterious effect in malignancy by inducing immunosuppression.
High Treg numbers have been detected systemically and
in the tumor microenvironment in patients with various
types of cancer and correlate with poor survival [8286].
Recently it has been demonstrated that an anti-PSCA-antiCD3 scDb not only effectively re-directed and activated
effector T-cells, but also Tregs resulting in an increased secretion of the immunosuppressive cytokine IL-10. Redirected
Tregs suppressed the proliferation and cytokine production of CD4+ effector T-cells both in vitro and in vivo
and abrogated the antitumor effector function of redirected Th -cells thereby promoting tumor growth in vivo
[87].
Despite these drawbacks, multiple studies have demonstrated that treatment with anti-TAA-anti-CD3 bsAbs can
result in the induction of an overall effective proinflammatory antitumor immune response and in clinical responses.
This may be explained by an initial recruitment of CD8+
effector T-cells, with CD4+ T-cells, including Tregs, being
recruited at a later stage [21,69]. Furthermore, tumor resident
T-cells were sufficient to induce substantial tumor reduction
in a NOD/SCID xenotransplanted mouse model treated with
BiTEs [69].
As of yet it remains unclear whether tumor targeting of
all CD3-expressing T-cells has significant clinical impact.
However, in order to maximize clinical effect and minimize
potential AE it seems more attractive to restrict tumor targeting to specific effector cell subsets with known antitumor
effects.

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Several specific immune effector cell subsets that

can be considered attractive candidates for tumor targeting (i.e. T-cells, invariant natural killer T-cells
(iNKT), and natural killer cells (NK)) are discussed
below.
5.1. T-cells
T-cells, once regarded an evolutionary redundant T-cell
subset en route to extinction, have been demonstrated to hold
a unique position in the immune system. They can directly
lyse stressed or infected cells, produce a diversified set of
cytokines and chemokines to regulate both immune and nonimmune cells, and can present antigens for T-cell priming
[88].
V9V2 T-cells constitute the predominant T-cell
subset in human peripheral blood. V9V2 T-cells can be
activated and expanded by non-peptidic pyrophosphate antigens (pAg), of which there are both host and microbe-derived
counterparts, typified by isopentenyl pyrophosphate (IPP)
and hydroxymethyl-but-2-enyl-pyrophosphate (HMBPP),
respectively. Furthermore, aminobisphosphonate compounds
(e.g. zoledronic acid) sensitize target cells to V9V2 Tcell killing by promoting the intracellular accumulation
of endogenous IPP by inhibiting mevalonate metabolism
[89,90]. Recently, it was reported that butyrophilin (BTN)
3A1 is required for the presentation of pAg to V9V2 T-cells
[91,92].
In stressed/malignant cells pAg production is frequently
upregulated allowing discrimination from normal tissue.
Indeed, V9V2 T-cells have been shown to be able
to recognize and eliminate malignant cells from multiple tumors types, including multiple myeloma (MM),
NHL, prostate-, renal cell- and colon cancer. Quantitative
and qualitative defects in the V9V2 T-cell population
are noted in various malignancies [90] and negatively
impact disease-free survival, e.g. in ovarian carcinoma
[93]. Importantly, these functional V9V2 T-cell defects
are reversible [90]. In patients with various metastatic
cancers treatment with zoledronic acid and IL-2 promoted the differentiation of peripheral blood V9V2
T-cells toward an effector/memory-like phenotype with
augmented numbers correlating with arrested disease progression. Observed toxicities were minor and limited to
transient flu-like symptoms [94,95]. In patients with various malignancies, adoptive transfer of V9V2 T-cells
following ex vivo expansion by pAg, aminobisphosphonates or mAbs combined with IL-2, resulted in clinical
responses [90]. Strikingly, in vitro V9V2 T-cell mediated lysis of hepatic tumor cell lines was significantly
enhanced by an anti-EpCAM-anti-CD3 BiTE [96]. Although
clinical data are scarce, preliminary findings clearly indicate that exploiting the natural abilities of V9V2 T-cells
in cancer immunotherapy is feasible and carries low
toxicity.

5.2. Invariant natural killer T-cells (iNKT)

iNKT represent a distinct population of lymphocytes
characterized by a (semi-)invariant TCR. Unlike conventional T-cells, iNKT recognize (glyco-)lipids presented by
non-polymorphic CD1d molecules and rapidly secrete a
wide range of cytokines upon stimulation thereby inducing
effector (e.g. NK and CTL) cell activation in an IFN-
dependent manner [9799].
iNKT contribute to immune surveillance in early-stage
tumors and chemically induced cancers and play a pivotal role in controlling different forms of cancer in mice
[100,101]. Moreover, iNKT from patients with advanced cancer display quantitative and qualitative defects and circulating
numbers correlate with patient survival [100103]. Reciprocal interactions between DC and iNKT can reverse defects
in the iNKT population. Indeed, in vitro results indicate
rehabilitation of iNKT function after stimulation with monocyte derived DC (moDC) pulsed with the agonistic CD1d
ligand -galactosylceramide (-GalCer) and exogenous IL12 [104,105]. It was shown that sustained activation of iNKT
at the tumor site could be induced after systemic treatment with -GalCer loaded soluble CD1d-molecules fused
to an anti-tumor scFv. Potent tumor inhibition of aggressive
tumor grafts expressing the targeted antigen was observed
in mice [106,107]. Although less clear than in mice, clinical
studies evaluating administration of -GalCer-pulsed moDC
with or without adoptive transfer of ex vivo expanded iNKT
have reported objective tumor regressions in several patients
[108112]. In patients with recurrent HNSCC, nasal submucosal administration of GalCer-pulsed APCs combined with
intra-arterial infusion of activated iNKT via tumor-feeding
arteries produced objective responses in 5 out of 10 patients.
The number of infiltrating iNKT in extirpated tumor tissue
correlated with clinical outcome [112].
In summary, extensive preclinical and early clinical data
underscore the important role of iNKT in tumor immunosurveillance and indicate beneficial effects with low toxicity
in cancer treatment, therefore redirecting this invariant subset
potentially constitutes a valuable approach.
5.3. Natural killer cells (NK)
NK cells represent a major subset of innate cytotoxic
lymphoid cells tightly regulated by inhibitory and activating signals sensed via cell surface receptors. Activation can
be triggered by a lack of inhibitory signals delivered by MHC
class I molecule engagement missing self via ligation of
the activating receptor NKG2D by stress-induced molecules
(e.g. MHC class I chain-related genes (MIC) A and B)
induced self and by FcRIIIA ligation (i.e. ADCC). Upon
activation, NK cells become highly cytotoxic (e.g. through
perforin- and granzyme mediated mechanisms) and secrete
pro-inflammatory cytokines (e.g. IFN-) [113,114].
Their role in tumor immunosurveillance is underscored by
epidemiological studies correlating disease prognosis with

R. Lameris et al. / Critical Reviews in Oncology/Hematology 92 (2014) 153165

tumor infiltrating NK cells and diminished cytotoxicity with

an increased risk of cancer development [114116]. Additionally, NK cells were shown to contribute to mAb based
cancer treatment though FcRIIIA (CD16A) mediated recognition and elimination of mAb coated tumor cells. ADCC
could be significantly enhanced through Fc-tail modifications resulting in increased FcRIIIA affinity, at least in vivo
[117].
Given the role NK cells play in first-line defense against
malignancies, their therapeutic use has been widely explored
in clinical trials. Adoptively transferred ex vivo expanded and
activated autologous NK cells proved to be safe, however no
significant clinical responses were observed. To break selftolerance associated with autologous NK, adoptive transfer
of allogeneic NK cells has been investigated as an alternative. An enhanced leukemia clearance rate was reported in
patients with poor-prognosis acute myeloid leukemia (AML)
with successful in vivo NK cell expansion after haploidentical related donor infusion and daily i.v. IL-2 [116,118]. Of
note, a substantial increase in host Tregs was reported after
combined IL-2 and NK cell therapy [119], possibly limiting
their therapeutic efficacy.
Though proven safe, NK cell therapy has thus far induced
modest and inconsistent clinical responses across various
cancer types and it has been argued that additional activation stimuli (e.g. FcRIIIa ligation) and/or homing to
tumors may be required for the induction of more robust
anti-tumor responses [116,118]. As noted, several bsAb constructs (e.g. Triomab, anti-CD19-anti-CD16 bsDb/TaFv and
an anti-CD33-anti-CD16 TaFv) are capable of enhancing
tumor destruction through effective FcR-mediated redirection and activation of NK cells [10,61,120]. Moreover,
dampened NK cell responses through tumor derived soluble
NKG2D-ligands were reversible upon cross-linking of tumor
and NK cells by an anti-CD30-anti-CD16A bsAb construct,
at least ex vivo [115]. Similarly, the fusion protein ULBP2anti-CEA scFv induced an effective anti-tumor response and
NK activation in a syngeneic colon carcinoma mouse model
[54]. Redirection of adoptively transferred NK cells by bsAbs
targeting tumor cells and NK cells may therefore prove to be
highly effective.
In conclusion, targeting invariant/conserved lymphocyte subsets through their highly conserved receptor (e.g.
V9V2-TCR, iNKT-TCR, NK-receptors or FcRIIIA)
allow for redirection of specific lymphocyte subsets with
intrinsic anti-tumor efficacy and can prevent the simultaneous
redirection of immunosuppressive T-cells. Importantly, their
conserved receptors preclude the need for individualized
treatment.

6. Concluding remarks
Advances in bsAb engineering have marked a new era
of Ab based cancer treatment and have resulted in an array
of constructs shown to be effective in inducing target cell

161

killing by engaged effector cells. Indeed, (pre-)clinical trials

have demonstrated results that are not just an enhancement
of those accomplished by conventional mAbs, but represent
a whole new therapeutic repertoire and embody a leap forward in terms of therapeutic efficacy. Nevertheless, further
improvements of the constructs with respect to increasing
their half-life, stability, and tumor penetration as well as
with respect to the specific immune effector cell subset to
be targeted are required.
Future research will reveal to what extent bsAbs will
impact cancer treatment, whether new formats such as VHH
can overcome shortcomings of existing constructs and what
impact redirection of different effector cell subsets will have
on clinical outcome. Undoubtedly, the expanding interest in
this field will result in multiple compounds entering clinical
trials in the near future shedding light on these issues.

Conict of interest statement

All authors have no conflicts of interest to declare.

Acknowledgments
This work is supported by grant no. 90700309 from The
Netherlands Organization for Health Research and Development (ZonMw), grant VU 2010-4728 from the Dutch Cancer
Society (KWF), and grant 14-0343 from the Association for
International Cancer Research (AICR).

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165

Biography
Hans J. van der Vliet is a medical oncologist at the
Department of Medical Oncology of the VU University Medical Center and Cancer Center Amsterdam. His translational
research focuses on cancer immunotherapy with a special emphasis on conserved immunoregulatory and immune
effector cell subsets.