Review TRENDS in Biotechnology
5 May 2004
A revival of bispecific antibodies
Peter Kufer1,2, Ralf Lutterbüse1 and Patrick A. Baeuerle1
1
Micromet AG, Staffelseestr. 2, 81477 Munich, Germany
2
Institute of Immunology, Goethestr. 31, 80336 Munich, Germany
Bispecific antibodies usually do not occur in nature but
are constructed by recombinant DNA or cell-fusion
technologies. Most are designed to recruit cytotoxic
effector cells of the immune system effectively against
pathogenic target cells. This complex task explains
why, after more than 15 years of extensive research,
many different formats of bispecific antibodies have
been developed but only a few have advanced to clini-
cal trials. Here, we give a brief history of bispecific anti-
bodies and review very recent progress towards
formats that are beginning to solve the major issues of
earlier formats. These improved bispecific antibodies
are expected to show clinical efficacy in patients with
cancer and other diseases, in a way that monoclonal
antibodies have shown in recent years.
Until now, the hybridoma technology invented by Köhler
and Milstein to generate monoclonal antibodies has
nourished the hope for therapeutic breakthroughs in
diseases with high medical needs not served sufficiently
by conventional therapies [1]. The hallmark of monoclonal
antibodies is their specific binding to a particular antigen,
which enables them to find their target precisely in vivo
while ignoring antigen-negative sites. Bound to a target,
therapeutic antibodies can deliver a toxic payload, act as
agonists or antagonists of receptors, or as neutralizers of
ligands. Antibodies might even bind many targets that are
not recognizable by small-molecule drugs.
Monoclonal antibodies of the IgG type contain two
identical antigen-binding arms and a constant fragment,
(Fc)g. The Fc part enables the antibody to function as an
adaptor protein, linking antibody-bound cells to immune
cells bearing Fcg receptors. Because there are different
Fcg receptors and other proteins binding to Fc portions of
antibodies, such as complement, monoclonal antibodies
can mediate multiple effects ranging from the recruitment
of immune effector functions to mere increase of serum
half-life by retention of IgG on non-signaling Fc receptors.
It was observed recently that human antibodies of the
IgG4 type can exchange their halves with each other,
potentially creating antibodies with dual specificity [2].
However, the biological relevance of this observation
remains obscure.
For treatment of malignant diseases, monoclonal
antibodies typically need to be modified to enhance efficacy
and to use them in humans. One important modification is
the reduction of immunogenicity of rodent monoclonal
antibodies by chimerization, humanization through
Corresponding author: Peter Kufer (peter.kufer@micromet.de).
www.sciencedirect.com 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.03.006
Vol.22 No.
grafting of complementarity determining regions (CDRs)
or using various technologies for recovery of fully human
antibodies, such as phage display libraries or transgenic
mice expressing human antibody repertoires. Reduced
immunogenicity of antibodies can prolong their half life
and, in the absence of a neutralizing immune response,
enable prolonged treatment. Another important modifi-
cation is arming the humanized antibody with additional
cytotoxic mechanisms, be it radioisotopes, bacterial toxins,
inflammatory cytokines, chemotherapeutics or prodrugs.
There is a growing number of approved cancer thera-
peutics that are efficacious either as chimerized antibody
(Rituximab [3]) or humanized IgG1 (Herceptin [4] and
Campath-1H [5]), or as conjugate with chemotherapeutics
(Mylotarg [6]) or a radioisotope (Zevalin [7] and Bexxar
[8]). In spite of this progress, the efficacy of monoclonal
antibodies for cancer treatment is still limited [9], leaving
great potential for further improvements. One class of
antibody derivatives with the promise of enhanced potency
for cancer treatment are bispecific antibodies.
From monoclonal to bispecific antibodies
Antibodies with a dual specificity in their binding arms
usually do not occur in nature and, therefore, had to be
crafted with the help of recombinant DNA or cell-fusion
technology. Among the first bispecific antibodies were
constructs designed to redirect T cells against cancer
target cells [10]. Target cells were killed when cytotoxic
T lymphocytes were tethered to tumor cells and simul-
taneously triggered by one arm of the bispecific antibody
that interacted with the T-cell receptor (TCR) – CD3
complex. The use of the monomorphic CD3 complex for
triggering T cells circumvented the restrictions of clono-
typic T-cell specificity and enabled a polyclonal cytotoxic
T lymphocyte response against target cells bearing the
antigen recognized by the second arm of the bispecific
antibody [11]. It is important to note that cytotoxic
T lymphocytes (CTL), which are considered to be the
most potent killer cells of the immune system, cannot be
engaged by monoclonal antibodies because they lack Fcg
receptors. Another development is bispecific antibodies
that simultaneously bind tumor cells and an activating
Fcg receptor, for example, CD64/FcgRI on monocytes [12].
Their binding to Fcg receptors can elicit effector cell
activation, without being competed by simultaneously
binding normal IgG.
Why so many bispecific antibody formats?
Production of bispecific antibodies in sufficient amounts
and purity was an obvious challenge from the beginning.
 Review TRENDS in Biotechnology Vol.22 No.5 May 2004 239

Although producing small amounts for in vitro studies and of a murine and a rat hybridoma cell line expressing
animal models was relatively straightforward, high and monoclonal antibodies with two IgG subclasses selected for
affordable production yields as needed for clinical grade their preferential pairing [16]. Bispecific antibodies from
material required major efforts. Thus, the intense parallel quadroma cell lines still closely resemble conventional
development of various molecular formats of bispecific monoclonal antibodies. Besides the dual-specific antigen
antibodies was largely driven by an attempt to solve binding fragment (Fab) parts, they contain an Fcg part and
production problems (Figure 1). can thus be considered ‘trispecific’. The additional inter-
The first approach to construct and produce bispecific action of such trispecific antibodies with Fcg receptors
antibodies was the quadroma technology [13– 15]. It is might have desired as well as undesired biological effects.
based on the somatic fusion of two different hybridoma cell To avoid undesired effects, bispecific F(ab0 )2 fragments
lines expressing murine monoclonal antibodies with the were prepared by removing the Fcg part through
desired specificities of the bispecific antibody. Because of enzymatic digestion [17]. Other approaches used chemical
the random pairing of two different Ig heavy and light conjugation of two different monoclonal antibodies or
chains within the resulting hybrid –hybridoma (or quad- smaller antibody fragments [18]. A straightforward
roma) cell line, up to ten different immunogloblin species method was the coupling of two parental antibodies with
are generated of which only one is the functional bispecific a hetero-bifunctional crosslinker, but the resulting prep-
antibody. The presence of mispaired by-products reduced arations of bispecific antibodies suffered from a significant
production yields significantly and required sophisticated molecular heterogeneity because reaction of the cross-
purification procedures. A major improvement to the linker with the parental antibodies was not site-directed.
conventional quadroma approach was the somatic fusion To obtain more homogeneous preparations of bispecific
antibodies two different Fab fragments have been chemi-
cally crosslinked at their hinge cysteine residues in a site-
(a) (b)
directed manner [19].
VH VH Recombinant DNA technology promised to overcome
CH1
shortcomings of conventional bispecific antibody pro-
Fab CH1
VL VL duction. Bispecific antibodies resembling the quadroma-
derived format but consisting of human instead of murine
CL CL and/or rat sequences could be designed by the elegant
S-S
‘knobs-into-holes’ strategy [20]. The mispairing of Ig heavy
CH2 chains was reduced in this technology by mutating
Fc selected amino acids forming the interface of the CH3
CH3 domains in human IgG. At positions within the CH3
domain at which the two heavy chains interact directly, an
Quadroma F(ab′)2 amino acid with a small side chain (hole) was introduced
into the sequence of one heavy chain and an amino acid
(c) (d) with a large side chain (knob) into that of the other one. As
VH a result, the more favorable protein interaction between
VL
CH1 knobs and holes led to the formation of up to 90% of the
VL correct bispecific human IgG by transfected mammalian
VH host cells.
CL Alternative recombinant strategies focused on smaller
Ck CH1
Jun Fos bispecific antibody constructs. A simpler structure and
smaller molecular size should enable bacterial expression
Heterodimeric scFv Heterodimeric Fab
of larger quantities in Escherichia coli. One strategy
made use of the natural ability of certain protein
(e) (f) domains to associate as heterodimers. The leucine
VH VL VL VH zipper domains of transcription factors Fos and Jun
were joined to the C terminus of two different single-
chain Fv antibody fragments [21], or, to the corresponding
VL VH VH VL heavy chain segments in a Fab-based approach [22].
However, expression in E. coli revealed preferential
Diabody Tandem scFv formation of stable Fos – Fos and Jun– Jun homodimers.
TRENDS in Biotechnology
Therefore, bispecific heterodimers had to be generated
finally in vitro in a second step by mixing the dissociated
Figure 1. Alternative bispecific antibody formats. (a) Quadroma-derived bispecific halves of homodimers. A similar approach relying on the
antibody, resembling monospecific monoclonal antibodies in structure and size. heterodimerization of the constant Ig domains Ck and CH1
(b –d) Heterodimeric bispecific antibodies of medium size, comprising constant
immunoglobulin regions and/or heterodimerization domains. (e– f) Bispecific anti-
led to the successful association of two different single-
bodies of minimal size, consisting only of variable immunoglobulin regions. chain Fv antibody fragments during periplasmic
Abbreviations: CH1 – 3, constant regions of Ig-heavy chain; CL, constant regions of expression in E. coli [23]. In contrast to the Fos– Jun
Ig-light chain; Fab, antigen binding fragment; Fc, constant fragment; VH, variable
region of Ig-heavy chain; VL, variable region of Ig-light chain. The difference in col- strategy, no additional in vitro-engineering steps were
ors (i.e. gray and purple) represents different binding specificities. required.
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240 Review TRENDS in Biotechnology
Recombinant DNA technology was also used to
generate particularly small bispecific antibodies consist-
ing of only two VH and two VL domains from two different
antibodies. In ‘diabodies’, each VL domain is connected by a
short peptide linker with the VH domain of the other
antigen specificity and vice versa [24]. The peptide linker
is three to 12 amino acids in length, which is too short to
enable single-chain Fv formation between non-cognate
V regions. Coexpression of respective VH –VL fusions in the
periplasm of E. coli resulted in stable bispecific diabodies.
A further development of diabodies are so-called tandem
diabodies [25]. Although diabody crystal structures
revealed considerable flexibility [26], the connection of
the antigen-binding arms by two amino acid linkers might
nevertheless cause steric restrictions. This is in line with
the observation that the antigen-binding arms in diabo-
dies with VL –VH configuration are more flexible than in
diabodies with VH – VL domain arrangement [27]. Thus,
diabodies in general are capable of bridging surface
antigens between different cells [28]; however, this
might become problematic in situations in which the
simultaneous accessibility of two antigens on two different
cells itself is restricted.
An alternative small format is the arrangement of two
single-chain antibody fragments (scFv) connected by a
flexible polypeptide linker on a single polypeptide chain.
First attempts to produce such tandem scFvs were made in
E. coli [29,30]. From these and more recent experiments
[31] it became clear that, in contrast to diabodies,
expression of functional bispecific single-chain antibodies
in E. coli was not feasible. Renaturation of denatured
protein from E. coli inclusion bodies was no solution either
as it resulted in poor yields of functional bispecific
antibody. A breakthrough for bispecific single-chain
antibodies came from their expression in mammalian
cells [32]. It seemed that the secretory pathway of higher
eukaryotic cells was capable of properly folding the four
consecutively aligned Ig domains, and secrete the tandem
scFv in decent yields as fully functional bispecific antibody
into the cell culture medium. Affinity purification by a
poly-histidine tag attached to the C terminus, and size-
exclusion chromatography enabled preparation of highly
homogeneous bispecific antibodies. The flexible Gly– Ser
linker connecting the two scFvs was chosen to be short to
prevent mispairing of non-cognate V regions, although
longer linkers turned out to work as well [32,33]. In
particular, there was no need for peptide linkers forming
secondary structures to keep the different antigen-binding
sites apart [34]. In contrast to diabodies, the two binding
sites in single-chain bispecific antibodies can rotate freely
and their axes can be kinked, which might facilitate
simultaneous binding of two antigen epitopes juxtaposed
on two different cell surfaces.
Biological effects of bispecific antibodies and clinical
experience
The vast majority of bispecific antibodies were designed to
redirect cytotoxic effector cells against target cells that
have a key function in disease processes (Figure 2). With
respect to target cells, most approaches selected anti-
bodies for bispecific antibody development recognizing
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Vol.22 No.5 May 2004
Target cell
Surface
antigen
Bispecific
antibody
Redirected
lysis
Triggering receptor
Cytotoxic
granules
Cytotoxic effector cell
TRENDS in Biotechnology
Figure 2. Redirected target-cell lysis by a cytotoxic effector cell, mediated by a
bispecific antibody. The simultaneous engagement by a bispecific antibody of
both a surface antigen on the target cell and a triggering receptor on the effector
cell induces the effector cell to kill the target cell by delivering a cytotoxic payload.
tumor-associated surface antigens to eliminate malignant
cells causing cancer. With respect to cytotoxic effector cells,
a variety of antibodies against triggering molecules of
cytotoxic effector cells were tested for bispecific antibody
development.
The best characterized triggering molecules for recruit-
ing myeloid effector cells are the high affinity IgG receptor
FcgRI/CD64 and the IgA receptor FcaRI/CD89. CD64 is an
activating receptor found on monocytes, macrophages and
dendritic cells and can be upregulated on neutrophils
by G-CSF, GM-CSF and IFN-g [35,36]. Monocyte-
mediated tumor-cell lysis by CD64-directed bispecific
antibodies required preactivation of the effector cells by
IFN-g and showed a dose – response relationship similar to
that found with antibody-dependent cellular cytotoxicity
(ADCC) induced by a humanized IgG1 antibody against
the same target [37]. Likewise, CD64-mediated recruit-
ment of neutrophils requires preactivation of the effector
cells [38,39]. So far, no consistent antitumor activity was
observed in clinical trials with the bispecific F(ab0 )2
antibodies MDX-210 (Her2/neu £ CD64), MDX-H210
( ¼ humanized MDX-210) and MDX-447 (EGFR £ CD64)
[40,41], although high doses (20 mg m22) were well
tolerated and biopsies of metastatic lesions showed
tumor localization of bispecific antibody [42]. The only
exception reported was a Phase II clinical trial in patients
with advanced Her2/neu-expressing prostate cancer trea-
ted with humanized MDX-H210 plus GM-CSF, in which
35% of the evaluable patients showed a prostate-specific
antigen (PSA) response [43]. Limited clinical efficacy
might be explained by preclinical data on MDX-210
showing that measurable tumor-cell lysis requires bispe-
cific antibody concentrations of 0.1 – 1 mg ml21 and high
effector-to-target (E:T) cell ratios of at least 40:1 even when
human neutrophils that had been prestimulated with IFN-g
and G-CSF were used [44].
CD89 is expressed primarily on neutrophils, monocytes,
macrophages and eosinophils [45]. In contrast to CD64,
expression of this activating receptor on neutrophils is
 Review TRENDS in Biotechnology Vol.22 No.5 May 2004
constitutive. Bispecific antibodies triggering CD89 could
be demonstrated to mediate redirected lysis of tumor cells
by freshly isolated neutrophils and monocytes without any
need for prestimulation [46]. To obtain acceptable killing
rates in vitro, however, high E:T ratios and concentrations
of bispecific antibody were required (Table 1). CD89-
directed bispecific antibodies have not yet been tested in
clinical trials.
Bispecific antibodies designed to redirect the cytotoxic
activity of lymphocytic effector cells against tumor targets
mainly engaged CD16/FcgRIII or CD3 as triggering
molecules. CD16 is the major activating receptor on
natural killer (NK) cells and mediates low affinity binding
of IgG. The hybrid –hybridoma-derived bispecific antibody
2B1 (Her2/neu £ CD16) showed few minor clinical
responses in a Phase I trial in patients with advanced
Her2/neu-overexpressing cancer [47]. Infusion of 2B1
induced significant increases in the levels of circulating
cytokines, and maximally tolerated dose (MTD) was
reached at 2.5 mg m22. The toxicity of the molecule has
been explained by its Fcg part leading to systemic
leukocyte activation by simultaneous engagement of
CD16 and other Fcg receptors [48]. Compared with 2B1,
recombinant bispecfic Her2/neu £ CD16 constructs in the
tandem scFv format devoid of the Fcg part, required a
100-fold higher concentration in vitro to achieve the same
peak tumor-cell lysis by peripheral blood lymphocytes
prestimulated with IL-2 [49]. Thus, the Fcg part is not only
responsible for clinical side effects but also contributes to
the biological activity of CD16-directed bispecific anti-
bodies. HRS-3/A9 is another CD16-directed bispecific
antibody already tested in clinical trials [50]. The
compound is a bispecific F(ab0 )2 antibody (without Fcg
part) targeting the CD30 antigen on Hodgkin and Reed-
Sternberg cells in patients with Hodgkin’s Disease (HD).
HRS-3/A9 was significantly less toxic than 2B1, and
maximum tolerated dose was not reached at 64 mg m22
when the first Phase I– II trial ran out of clinical test
material because of the limitations of the production
process. The first study reported one complete remission
(CR) and one partial remission (PR) in 15 treated patients
[51], which could be confirmed by 25% objective antitumor
responses (1 CR þ 3 PR) in a second clinical trial [52].
Therefore, HRS-3/A9 can be regarded as the first clinical
proof of the bispecific antibody concept, although the
problem of insufficient drug supply remains unresolved.
CD3 is chosen as the triggering molecule in most
bispecific antibody approaches aimed at redirecting
cytotoxic T-cell activity against tumor cells. CD3 is a
T-cell-specific complex of three different monomorphic
Table 1. Characteristics of bispecific antibodies recruiting cytotoxic effector cells and requirements for redirected target-cell lysis
Triggering molecule CD64 CD89
Bispecific antibody format F(ab0 )2 F(ab0 )2
Effector cells Monocytes and neutrophils
Requirement of pre- or costimulation Yes No
ED50 range [mg ml21] 0.1– 1 0.1 –1
E:T ratio $ 40:1 100:1 –200:1
Refs [44] [46]
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241
chains (1, g and d) associated as signal transduction units
with the polymorphic TCR, which is devoid of signalling
properties. Thus, CD3 engagement by bispecific antibodies
can mediate TCR signaling by circumventing the clono-
typic antigen specificity of T cells. However, stimulation of
T-cell activity is a complex process strictly regulated by
several molecules, such as the costimulatory molecule
CD28. Indeed, most CD3-directed bispecific antibodies
require costimulation or prestimulation of T cells to elicit
cytotoxic activity against target (tumor) cells. This
dependency has been observed in vitro and in animal
models and applies to the conventional bispecific F(ab0 )2
format as well as to various recombinant diabodies [28,
53 – 58]. Most T-cell-redirecting bispecific antibodies
require an excess of effector cells for significant tumor-
cell lysis (E:T ratios of at least 2:1), and a broad range of
bispecific antibody concentrations is reported in the
literature for efficient induction of redirected T-cell
cytotoxicity (Table 1). So far, indications for clinical efficacy
of CD3-directed bispecific antibodies have come mainly
from locoregional or intraperitoneal treatment of cancer
patients [59 – 62], or from adoptive transfer of T cells coated
ex vivo with bispecific antibody [63,64]. However, systemic
application, which is the desired route for bispecific
antibody therapy of, for instance, metastatic cancer, has
not shown clinical efficacy up to now. In one study, patients
with B-cell malignancies were treated by intravenous
infusion of a hybrid –hybridoma-derived bispecific anti-
body against CD19 and CD3 with or without IL-2 [65].
Doses of up to 5 mg were administered, which caused a
massive systemic release of cytokines but no clinical
responses. Another Phase I clinical trial in renal cell
cancer patients receiving bispecific F(ab0 )2 antibody
against Ep-CAM, CD3 and IL-2 also revealed systemic
cytokine release, which, in this case, was dose-limiting at
5 mg kg21 [66]. No antitumor responses could be observed
at the tested dose levels.
In addition to Fc receptors and the TCR –CD3 complex,
several other, less potent triggering molecules such as
CD2, which is present on T and NK cells [67] were
evaluated for effector cell recruitment through bispecific
antibodies [68].
Except for the recruitment of immune effector cells,
bispecific antibodies have been designed for clearing
pathogens from the bloodstream through specific targeting
to CR1 (complement receptor 1) on erythrocytes [69,70] and
for specific interference with complement regulators on
tumor cells [71]. Moreover, bispecific antibodies can be used
for the specific targeting of toxins [72], chemotherapeutic
drugs [73] and radioisotopes to tumor cells [74]. In yet
CD16 CD3
Quadroma
F(ab0 )2 F(ab0 )2 BiTE
Tandem scFv Diabody
NK cells T cells
Yes Yes No
0.03 –1 0.001 –1 1025 –1024
50:1 $ 2:1 # 1:10
[49] [28,57] [80]
242 Review TRENDS in Biotechnology
another approach, bispecific antibodies are used to target
two different tumor-associated antigens simultaneously, to
achieve enhanced and more specific tumor targeting [75].
BiTEs: a promising new class of bispecific antibodies
Several factors limit the biological activity of most tumor-
directed bispecific antibodies. Even those bispecific anti-
bodies triggering the most potent activating receptors on
respective effector cells, i.e. CD64 or CD89 on monocytes
and neutrophils, CD16 on NK cells, and CD3 on T cells,
suffer from at least one of the following shortcomings:
(i) the need for additional signals inducing expression of
the triggering receptor and/or a pre- or costimulation of
effector cells; (ii) high concentrations of bispecific antibody
for decent target-cell lysis, which cannot be realized in
patients because of limited drug supply; (iii) dose-limiting
toxicity, and (iv) an excess of effector over target cells,
which is rarely encountered in vivo (Table 1).
It now seems possible to overcome most if not all
previous limitations by a new class of bispecific antibodies,
designated ‘bispecific T-cell engager’ (BiTEs). BiTEs are
recombinant bispecific single-chain antibodies consisting
of two scFv antibody fragments directed against a surface
antigen on target cells and CD3 on T cells. BiTEs can be
efficiently produced as fully functional molecules by
mammalian cells. In contrast to other CD3-directed
bispecific antibodies, BiTEs are capable of efficiently
redirecting T-cell cytotoxicity against various different
target cells without any requirement for pre- or costimula-
tion of effector T cells [32,76– 78]. This might be explained
by their ability to induce immunological cytolytic synapses
between target cells and cytotoxic T cells that are
indistinguishable in composition, subdomain arrange-
ment and size from synapses induced by regular T-cell
stimuli (S. Offner et al., unpublished). Typically, engage-
ment of only a few TCR molecules per T cell is sufficient to
trigger synapse formation and T-cell cytotoxicity efficiently
[79]. Likewise, target cells obviously need to be decorated
with only a few BiTE molecules to be killed specifically by
T cells. This is in line with the observation that BiTE
concentrations as low as 10 –100 pg ml21 (, 0.2 –2 pM) are
usually sufficient for half-maximal target-cell lysis [80].
Sub-microgram amounts of a BiTE were also sufficient to
prevent tumor outgrowth in a mouse model [81]. Data from
video-assisted microscopy show that BiTEs mediate serial
killing of many target cells by single cytotoxic T cells
(P. Hoffmann et al., unpublished), explaining how BiTEs
can lead to a complete lysis of target cells at E:T ratios as
low as 1:10 [82]. Obviously, cytotoxic activity at particu-
larly low E:T ratios is one of the most important
prerequisites for clinical efficacy, which has not been met
by any other bispecific antibody format so far. Moreover, it
is an attractive feature of BiTEs that no coadministration
of costimulatory agents is necessary that would otherwise
inevitably result in demanding multistep or multicom-
pound treatment regimens.
Future perspectives
Therapeutics based on bispecific antibodies have not yet
yielded the anticipated clinical success. However, this
review shows that further developments are ongoing with
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Vol.22 No.5 May 2004
considerable pace and ingenuity. Bispecific formats that
are still actively pursued in preclinical and early clinical
development include diabodies and tandem diabodies,
crosslinked F(ab0 )2, trispecific quadroma antibodies and
single-chain bispecifics. Their success in the clinic will
hinge on the quality of the target selected for recognizing
cancer cells, whether large-scale production is feasible,
and on the biological activities of recruited effector cell
population.
The critical selection of the antigen for tumor targeting
is a challenge shared with all other antibody-based
approaches exploiting the exquisite specificity of anti-
bodies. Antibodies against a great variety of tumor-
associated antigens have been characterized over the
past 25 years. Those that have shown good tumor imaging
and an acceptable safety profile in the clinic are good
candidates for an enhancement of efficacy through
conversion into a bispecific format that is expected to
have a higher potency and a particular benefit through
using ‘natural’ immune effector mechanisms.
Complexity of production and cost of goods is a
permanent issue with biologicals and, in particular, with
antibody derivatives. Simplicity and robustness of a
bispecific construct, reduction of the number of production
steps, and use of powerful production systems are key
parameters to solve the production issues that have
dominated the field of bispecific antibodies for a long time.
The race is still open as to which effector cell population
of the immune system, after recruitment through bispe-
cific antibodies, is best suited to eliminate tumor cells. The
increasing list of immune evasion mechanisms discovered
in late-stage tumor cells [83] strongly suggests that T cells
are the worst enemies of tumor cells. MHC-class-I-
negative tumors, which are no longer recognized by
T cells, are frequent in the metastatic situation [84]. In
earlier stages – in which tumors are smaller, genetically
more homogenous and better penetrable by antibodies –
other immune effector cells bearing CD16 or CD64 as
targets might have good therapeutic effects when
recruited by bispecific antibodies. Much more has to be
learned about the potential of the various immune effector
cells recruited by bispecific antibodies for cancer treat-
ment. Because the bispecific antibody essentially works as
a catalyst, repeatedly tethering a complex toxic payload –
namely an entire immune effector cell – to the cancer cell,
it is vital to understand what immune cells can achieve at
which stage of cancer after being recruited by a bispecific
antibody. The number of immune cells in the periphery
and target tissue, their state of activity, cytotoxic potential,
ability to penetrate (tumor) tissue, ability to effect serial
killing, and susceptibility to evasion mechanisms of
tumors are just a few of the parameters that must be
studied in this respect.
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