Beruflich Dokumente
Kultur Dokumente
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
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.
154
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
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.
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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.)
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
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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
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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
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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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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.