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Current Opinion in Drug Discovery & Development 2009 12(2):

© Thomson Reuters (Scientific) Ltd ISSN 1367-6733

Bispecific antibodies for cancer therapy

Patrick Chames* & Daniel Baty

Address

Institut de Biologie Structurale et Microbiologie, Laboratoire d'Ingénierie des Systèmes Macromoléculaires,

CNRS UPR 9027, GDR 2352, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

Email: pchames@ibsm.cnrs-mrs.fr

*To whom correspondence should be addressed

The 2 last decades have seen the emergence of monoclonal antibodies as therapeutics. Nine monoclonal

antibodies have been approved for cancer therapy. However the efficiency of these molecules is far from

optimal, and antibody engineering is actively used to improve them. Because of their ability to simultaneously

bind two different antigens, bispecific antibodies are unique, and their wide potential as retargeting reagent has

been demonstrated over the years. However their use as therapeutics has been restrained by manufacturing

challenges. Several new recombinant formats have changed the situation. Innovative molecules have led to

impressive preclinical and clinical results, and hold great promise. This review presents an overview of the most

promising candidates.

Keywords Bispecific, cancer therapy, immunotherapy, retargeting, single domain antibodies

Abbreviations

ADCC antibody-dependent cell-mediated cytotoxicity, bsAb bispecific antibody, BiTE bispecific T-cell engager,

dAb domain antibody, Db diabody, DC dendritic cell, FcR Fc receptor, IFN interferon, Ig immunoglobulin, IL

interleukin, PBMC peripheral blood mononuclear cell, scFv single chain Fv, TaFv tandem scFv

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Introduction

Monoclonal antibodies (mAbs) are endowed with exquisite specificities, and since their discovery in 1975, they

have raised many expectations for the development of novel treatments, particularly in cancer therapy [1].

However, all of these molecules had to be extensively optimized and engineered before they were able to

deliver approved therapeutic molecules [2]. Nine mAbs are currently approved for cancer therapy, and

hundreds are at different stages of clinical development [3]. Despite the enthusiasm for developing mAbs for

therapy, most mAbs show limited efficacy, and so there is great potential for further improvement in this

research area. Most mAbs function either by directly blocking a ligand or a receptor, sometimes leading to

apoptosis, or as an adaptor molecule capable of recruiting effector cells from the immune system through

interaction between their Fc portion and various Fc receptor (FcR) bearing cells. Consequently, considerable

efforts are spent in the optimization of the Fc/FcR interaction [4].

Bispecific antibodies: Concepts, formats and limitations

However, as hypothesized in 1985, one solution for many mAb shortcomings might be the creation of bispecific

antibodies (bsAbs), which would be capable of the simultaneous binding of two different targets and thus also

capable of delivering a great variety of payloads to cancer cells [5]. BsAbs, however, do not exist in nature.

Despite numerous attempts and various proposed formats, the production BsAbs in significant quantities

remains, to a certain extent, a challenge. Three main approaches to the large-scale production of bsAbs have

been described. The first was the quadroma technology, involving a somatic fusion of two different hybridoma

cell lines [6]. Because of the random pairing of the two heavy and two lights chains of the antibody, the use of

sophisticated purification procedures to isolate the bsAb was required. This inefficient, time- and resource-

consuming approach led to the production of small amounts of bsAb that, although were sufficient for

characterization, were insufficient for clinical requirements. Numerous studies have demonstrated the potential

of such bsAbs but have also highlighted important shortcomings, including the high immunogenicity of such

murine molecules and a high toxicity, which was attributed to the presence of an Fc portion capable of

stimulating various FcR-bearing cells [7]. As a consequence, quadromas are no longer favored, with one

notable exception: Lindhofer et al demonstrated that the fusion of a murine immunoglobulin (Ig)G2a-producing

hybridoma and a rat IgG2b-producing hybridoma may lead to preferential heavy/light chain pairing and easy

separation of the homo- and heterodimers of heavy chains [8]. This approach can thus efficiently produce

bispecific forms of whole IgG. Moreover, the resulting hybrid Fc portion of the resulting antibody has been

shown to efficiently bind to activating human FcRs [9]. These molecules, called triomabs, are thus considered to

be trispecific, and have produced exciting results in clinical trials (see below) [10].

The second approach that was developed to produce bsAbs is based on the use of heterobifunctional chemical

reagents. The three major problems associated with chemical cross-linking are: (i) product homogeneity,

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leading to poor yields and complicated purification steps; (ii) possible inactivation of the molecules or poor

stability; and (iii) toxicity associated with the presence of Fc portions. Nevertheless, such molecules, such as

CD20Bi and Her2Bi, have been evaluated in the clinic [11]. To decrease toxicity, F(ab') can be produced by

enzymatic digestion and mild reduction of the two parental IgGs, followed by heterodimerization via thiol-

reactive heterobifunctional reagents. Such artificial bispecific F(ab')2 have been extensively studied, and some

molecules have reached the clinic, although none of them have been approved so far. [12].

The implementation of recombinant antibody techniques has revolutionized the bsAb field, leading to a third

approach. More precisely the ability to use single-chain FV (scFv) fragments (antibody variable domains linked

by a flexible peptide linker) has been a source of inspiration [13]. Two formats have been intensively studied.

Tandem scFvs (TaFv) consist of two scFv fragments linked via an extra peptide linker. This 50-kDa molecule,

which is difficult to produce in Escherichia coli, is well expressed by mammalian cells and is expected to confer

a good flexibility to each scFv fragment. This format has been used to create bispecific T-cell engager molecules

(BiTE), which are extremely potent bsAbs that have shown impressive results in clinical trials [14].

By reducing the length of the peptide linker between variable domains so that the domains cannot assemble, it

is possible to force the pairing of domains from two different polypeptides, leading to compact bsAbs called

diabodies (Dbs) [15]. These molecules can be expressed at high yields in bacteria, and have been shown by

crystallography experiments to adopt several conformations [16]. The Db format has been improved further by

the addition of an extra peptide linker between the two polypeptides in order to further decrease the number of

homodimers formed, leading to a fragment called a single chain diabody (scDb) [1]. Numerous studies have

demonstrated the potency of these formats, although the reduced flexibility of the two binding sites might

prove to be a limitation in some cases.

In addition to TaFv and Db, several other scFv-based formats have been proposed which use various

heterodimerization motifs including the human CH1/Cκ domains and CH3 domain pairs that have been elegantly

engineered to favor heterodimerization [17]. More recently, an approach called Dock-and-Lock was used to

create trivalent bispecific molecules [18]; this method relies on the natural interaction between the 44 C-

terminal residues of the regulatory unit of human protein kinase A, which spontaneously dimerize and bind to a

17-residue motif derived from the A-kinase anchoring protein. This trimerization motif has been stabilized by

the introduction of cysteine residues into both domains, leading to the formation of covalent disulfide bonds.

Such 150-kDa trivalent molecules have been successfully used for pretargeting strategies (see below) [18].

Recently, a new format consisting of fusions of an extra variable domain to each N-terminus of an IgG (ie, VH1-

VH2-CH1-Fc and VL1-VL2-CL) was described [19]. The resulting bispecific molecules, called dual-variable-

domain antibodies, have been shown to fold correctly and to exhibit pharmacokinetics similar to those of the

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parental mAbs [19]. Steric constraints have to be expected for one of the binding sites, although this antibody

format has been shown to successfully target soluble targets.

Finally, recent years have seen the emergence of domain antibodies (dAbs), which were first engineered from

mouse VH domains [20], and later from human variable domains [21]. DAbs are also found in nature in some

isotypes of camelids and sharks [22,23]. These fragments are compact, extremely stable, and do not need

domain pairing, and so represent ideal building units for the generation of more complex molecules including

multispecific or multivalent molecules. For example, bispecific tandem dAbs can be produced efficiently using

flexible linkers and such molecules have demonstrated excellent production yields and stability [24].

Interestingly, their modularity has been used to create a linker-free bsAb by directly fusing two llama-derived

dAbs to a human CH1/Cκ heterodimerization motif, thereby creating a Fab-like bispecific antibody fragment

termed bsFab [Cornillon, A. et al, unpublished]. These bispecific molecules are easily produced, extremely

stable, and do not suffer from the usual limitations experienced by linker-based molecules, including production

issues, poor stability, aggregation that can favor immunogenicity, and proteolytic degradation.

Pharmacokinetics of bispecific antibodies

Several of the methods for producing bsAbs described above generate bispecific antibody fragments of a small

size (eg, 30 kDa for TaFv). Although a small size is clearly an advantage in terms of tumor penetration, it is

also a potential problem because small molecules administered through intravenous injection are rapidly

eliminated from the circulation by renal clearance. Moreover, several bsAb formats do not possess an Fc

portion, and thus do not bind to the neonatal Fc receptor (FcRn), which is a receptor expressed by endothelial

cells and responsible for the very long serum half-life of IgGs [25].

Several solutions have been designed to overcome the rapid clearance of of bsAbs. Addition of PEG to bsAbs

has been shown to improve the serum half-life [26], but this often alters the binding affinities of bsAbs [27],

which might affect tumor targeting. Two other methodologies designed to reduce the clearance of bsAbs are

based on the interaction between human serum albumin (HSA) and FcRn. HSA, as is the case with IgG, enables

recycling of the bsAb by endocytosis, thereby leading to reduced renal clearance and extended serum half-life.

Indeed, a direct fusion of a bsAb to HSA can significantly improve its kinetics, as has been demonstrated for

anti-CEA x CD3 Dbs [28]. The fusion of a Fab fragment to HSA-binding peptides has been shown to increase

the serum half-life of the Fab fragment by 10- to 15-fold, leading to superior in vivo efficacy of the fragment

compared to the full IgG [29]. This general strategy has not yet been applied to bsAb but should be applicable

with success to TaFv and Db. Similarly, small dAbs (llama and human) that bind strongly to serum albumin

have been isolated. These dAbs have an extended serum half-life matching that of serum albumin itself [30],

and can be fused to other antibody fragments to create bsAbs with favorable kinetics [31].

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New strategies enabled by bispecific antibodies

(Sub) Dual targeting

The inhibition of signaling pathways is one of the main modes of action of therapeutic antibodies. Combinations

of small-molecule-based treatments can lead to additive or even synergistic effects [32], block redundant

signaling pathways and minimize the possibility of escape from therapy, but are often not adopted in the clinic

because of issues with high toxicity. However, human or humanized mAbs are often very well tolerated and are

thus very good candidates for combination therapies. Consequently, several companies are actively testing

combinations of mAbs, including Genentech Inc with trastuzumab (anti-Her2) plus bevacizumab (anti-VEGF),

and Immunomedics Inc with epratuzumab (anti-CD22) plus rituximab (anti-CD20) [33]. However, the

combination of therapeutic antibodies faces several limitations, including intellectual property issues and the

high costs involved in research, manufacturing and regulatory affairs. An elegant answer to these difficulties

might be the simultaneous inactivation of two targets by a single (bispecific) antibody, as exemplified by two

studies [34,35] that successfully simultaneously targeted epidermal growth factor (EGF) and insulin-like growth

factor (IGF) receptors, demonstrating that simultaneous targeting is more efficient than monotherapies against

the same targets.

(Sub) T-cell retargeting

Perhaps the most obvious applications of bsAb are in T-cell retargeting. Cytotoxic T-cells are considered the

most potent killer cells in the immune system. These T-cells are abundant, can efficiently proliferate upon

activation, can kill multiple times [36], and can efficiently infiltrate tumors but do not express Fcγ receptors.

The concept of applying the novel bsAb methodology to enable T-cells to kill tumor cells more potently emerged

in the 1980s [5]. BsAbs directed against both a tumor marker and CD3 have the potential to redirect and

activate any circulating T-cells against tumor cells. However, T-cells have a major drawback. Without a

secondary signal provided by the interaction between CD28 and one of its ligands, such as B7, T-cells are not

fully activated and may even become anergic [37]. The first anti-CD3 bsAbs were therefore applied in

combination with anti-CD28 antibodies, leading to mixed results [38]. Other alternatives are being explored,

such as the massive ex vivo expansion (> 300 x 109) and activation of patient's T-cells using low concentrations

of anti-CD3 and interleukin (IL)-2, followed by reinjection of these polyclonally activated T-cells decorated with

an anti-CD3 bsAb [11]. Interestingly, activated T-cells armed with Her2/neu bsAbs have been demonstrated to

be resistant to activation-induced cell death and are able to proliferate and kill tumor cells repeatedly [39].

Surprisingly, a small recombinant bsAb format appears to bypass the need for costimulation. Baeuerle and

collaborators are developing a murine TaFv BiTE [40]. An anti-CD19 x CD3 BiTE exhibited very efficient

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inhibition of tumor growth in subcutaneous leukemic human B-cell lymphoma xenograft models at low

concentrations and low effector-to-target ratios [41]. Similar impressive results, including the eradication of

tumors from both a human colon cancer cell line and ovarian cancer metastasis in immunodeficient mice, have

been obtained with an anti-EpCAM x CD3 BiTE [42]. In immunocompetent mice, a BiTE directed against the

murine CD3 led to a strong antitumor effect in the absence of costimulation [43]. The proposed explanation for

the outstanding efficacy of this BiTE in the absence of costimulation involves the highly efficient formation of

immunologic synapses due to the very short linker (five residues) connecting the two scFv fragments [44]. In

the last year, the significant potential of these molecules has been clearly demonstrated in clinical trials [14].

Blinatumomab (MT-103, Micromet Inc/MedImmune), an anti-CD3 x CD19 BiTE, was shown to induce partial and

complete regression in non–Hodgkin's lymphoma patients at extremely low doses (15 µg/m²/day) equating to

approximately five orders of magnitude below the serum levels reported for efficacious amounts of rituximab

(375 mg/m²/week). All of the seven patients treated with 60 µg/m²/day of blinatumomab experienced tumor

regression. A clinical trial targeting solid tumors has also been initiated by Micromet with another BiTE, MT-110

(anti-EpCAM x CD3) [45].

However, T-cell stimulation via CD28 has been shown to be highly efficient and self sufficient. rM28, a TaFv

dimer directed to a melanoma-associated proteoglycan (NG2) and to the costimulatory CD28 molecule on

human T-cells induced pronounced T-cell activation in peripheral blood mononuclear cell (PBMC) preparations

without additional T-cell receptor (TCR)/CD3 stimulation being required via supra-agonistic T-cell activation.

Interestingly, cytokines produced by CD28-stimulated T-cells were demonstrated to activate natural killer (NK)

cells, which subsequently significantly contribute to tumor cell lysis [46,47]. The safety and efficiency of

intralesional application of the murine bispecific TaFv rM28 and autologous PBMCs in patients with stage III/IV

metastatic melanoma and unresectable metastasis are currently being studied in a phase I/II clinical trial.

Perhaps the most unexpected bsAb successes have been the rat/mouse chimeric full IgG anti-CD3 bsAbs, which

are named triomabs and were designed by Lindhofer and collaborators. The resulting chimeric Fc portion of the

triomabs was shown to efficiently interact with activating human FcRs (FcγRI and FcγRIII) but not inhibitory

FcRs (FcγRIIB), thereby achieving the goal of several academic and industrial laboratories using human Fc

engineering. Furthermore, triomabs were shown to be capable of activating dendritic cells (DCs), inducing NK-

dependent antibody-dependent cell-mediated cytotoxicity (ADCC), and stimulating tumor cell phagocytosis by

macrophages [9,48]. Consequently, the chimeric Fc adds two crucial functions to the anti-CD3 x target bsAb:

additive tumor killing capabilities through the recruitment of macrophages and NK cells, and, most importantly,

the efficient costimulation of T-cells through direct contact with accessory cells such as macrophages and DCs

(B7/CD28, CD40/CD40L, LFA3/CD2) or cytokine secretion (IL-2, IL-6, IL-12). The only limitation of these

exciting bsAb molecules resides in their immunogenicity, which precludes repetitive injection of these bsAbs in

large quantities. Nevertheless, triomabs have exhibited impressive preclinical and clinical results, with

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reductions of up to 5 log in the number of tumor cells in ovarian carcinoma patients, despite the use of low

doses of bsAb to prevent reversible adverse effects [49]. Three of these chimeric bsAb molecules,

catumaxomab (anti EpCAM x CD3), ertumaxomab (anti Her2 x CD3) and FBT-A05 (Lymphomun; anti CD20 x

CD3), are under development by Fresenius Biotech GmbH and under preregistration (catumaxomab) or in

phase II clinical trials (phase both ertumaxomab and FBT-A05).

(Sub) FcγR-bearing cell retargeting

Although T-cells are very attractive effector cells, NK cells have also been shown to be very efficient at

destroying tumor cells. ADCC is thought to be one of the main modes of action of therapeutic antibodies, as has

been demonstrated by the association of functionally relevant FcγRIIIA polymorphisms with clinical responses to

rituximab [50]. NK cells are known to mediate essentially all of the ADCC measured in vitro, using PBMCs as

effector cells. Of note is the fact that neutrophils, the most numerous leukocyte type, express FcγRIIIB (which is

extremely homologous to FcγRIIIA), but cannot be activated to induce ADCC through this receptor. In vitro,

neutrophils do not interfere with ADCC efficiency [51]. NK cells have the advantage over T-cells in that pre-

activation is not required, because NK cells constitutively exhibit cytolytic functions [52]. However, NK cells

represent less than 10% of lymphocytes, and very small numbers of NK cells are found in direct contact with

tumor cells, suggesting that NK cells poorly infiltrate tumors [13]. Many ongoing studies aim to optimize NK cell

killing through engineering of the Fc/FcγRIIIA interaction [4]. Indeed, therapeutic antibodies suffer from several

limitations, not only the influence of FcγR polymorphisms as described earlier, but also the influence of the

glycans that are post-translationally added to the IgG CH2 domain, competition with high concentrations of

circulating IgG for binding to FcγRIIIA, and co-activation of inhibitory receptors such as FcγRIIB decreasing the

overall immune response [2]. As postulated in the early 1990s, one way to overcome all of these limitations

would be to redirect and activate NK cells using anti-target x FcγRIIIA bispecific antibodies that targeted an

epitope not involved in IgG binding [53,54]. The potential of this approach has been validated in clinical trials

[55,56]. However, significant human anti-mouse responses prevented further clinical evaluation of these

quadromas. One of these promising bsAbs (HRS-3/A9) was converted to a bispecific Db format, and the

resulting molecule was shown to induce tumor lysis by NK cells more efficiently than the parental molecules

[52], and is expected be less immunogenic than quadroma derived because of the absence of the Fc portion.

Yet this antibody fragment still carries the framework sequences of murine origin. The clinical potential of bsAb-

based NK cell retargeting still has to be demonstrated. For this goal, the authors of this article have isolated

activating anti-FcγRIIIA dAbs [57] and anti-CEA dAbs [Behar G et al, unpublished results] that have been used

to produce anti-CEA x FcγRIIIA bsFab molecules. These stable and efficiently produced bsAbs, capable of

inducing very potent ADCC at sub-picomolar concentrations [Cornillon et al, unpublished results], are currently

undergoing preclinical studies.

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A third class of interesting effector cells is the neutrophils. These cells are of special interest because several

studies support their in vitro and in vivo role in tumor cell lysis and tumor rejection [58-61]. Moreover,

neutrophils represent by far the most abundant FcR-expressing leukocyte subset in blood, and their numbers

can be increased by treatment with granulocyte colony-stimulating factor (G-CSF). FcαRI is constitutively

expressed on myeloid effector cells, including neutrophils, monocytes, macrophages, eosinophils and DCs, but

not on non-cytolytic cell populations [62]. Because FcαRI is capable of efficiently activating immature

neutrophils [63], targeting this receptor by the use of bsAbs has considerable potential that should be explored

more extensively in the future.

Finally, FcγRI (CD64) has also been targeted by bsAbs. This receptor is constitutively expressed by monocytes

and macrophages. Moreover, the expression of FcγRI can be induced at the surface of neutrophils upon

stimulation with interferon (IFN)γ or G-CSF. Most notably, two bsAbs, constructed by the chemical conjugation

of Fab' fragments, have been extensively studied for the treatment of advanced breast cancer (anti-Her2 x

CD64) and head and neck cancer (anti-EGFR x CD64). Both of these bsAbs were tested in phase I clinical trials

[12]. The molecules were well tolerated, but unfortunately did not lead to consistent antitumor activity. The

lack of efficacy was suggested to be due to both an effector-to-target ratio and a bsAb concentration that were

too low. An anti-EpCAM x CD64 bsAb also demonstrated efficient carcinoma cell killing using G-CSF- and IFNγ-

stimulated neutrophils [64], supporting the concept that activated neutrophils might represent efficient tumor

killing cells.

(Sub) Pretargeting

Radioimmunotherapy approaches have several advantages over more conventional treatments for cancer. One

of the most attractive advantages is the bystander effect. In a particular tumor, cells often tend to lose antigen

expression to escape from therapy. However, radiolabeled antibodies bind to antigen-positive tumor cells and

can irradiate cells in their close proximity (bystander cells), including antigen-negative tumor cells, tumor

stromal fibroblasts and tumor vasculature. Unfortunately, and despite promising results obtained during the last

25 years with directly labeled antibodies, few convincing positive clinical results have been achieved, mostly

because IgG remains in the circulation for days and causes unacceptable toxicities to normal tissues [65].

Almost 20 years ago one method proposed to resolve this problem involved the separation of the antibody

targeting moiety from the radiolabeled effector [66]. Antibody fragments rapidly clear from the circulation and

often show better tumor penetration and faster maximum tumor uptake than whole antibodies, but overall lead

to very poor total tumor uptake and an insufficient radiation dose. In the two-step pretargeting method, a bsAb

targeting the tumor would be injected in the first step. Once optimal tumor uptake and clearance of the bsAb

from the circulation had occurred, the second step would involve the injection of a small radiolabeled effector

that would be captured at the tumor site by the bsAb and rapidly cleared from the rest of the system, thereby

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sparing the organism from excessive doses of radiation. Although the potential of the bsAb pretargeting

technology was shown to be successful in both preclinical and clinical experiments, the poor production yield

caused by the use of chemical constructs has so far limited the expansion of this approach as a clinically

available therapy [67].

Using the Dock-and-Lock approach, Goldenberg and collaborators have produced trivalent bsAbs, which are

composed of two anti-MUC1 Fabs linked to Fab TF2 binding to the hapten HSG (histamine-succinyl-glycine).

This molecule was used in the pretargeting of 90Y-labeled hapten, and led to ratios of more than 100 to 1 in

tumor to non-tumor normal tissue and average tumor to blood ratios of 1000 to 1 in animal model studies. A

single injection of this molecule could potentially lead to complete regression of eight out of ten tumors,

demonstrating the high clinical potential of this efficiently produced bsAb [18].

The Holy Grail: BsAb-dependent induction of adaptive response?

As demonstrated by successful clinical trials, the possibility of efficiently retargeting effector cells to destroy

tumor cells seems to be within reach. However, if bsAb-based therapies could also be developed to stimulate

the involvement of the adaptive immune system, tumor killing T-lymphocytes could potentially be generated,

and vaccination could be established (ie, the provision of long-term immune response and memory cells that

could rapidly generate a new and efficient response in the case of metastasis development). The induction of an

adaptive response would be dependent on the uptake of tumor cell debris resulting from bsAb-dependent lysis,

and presentation of the debris by macrophages, B-cells and DCs, as complexes with major histocompatibility

complex (MHC) molecules. However, DCs normally present exogenous antigen through MHC class II molecules

whereas cytotoxic T-cell activation requires presentation through MHC class I molecules. Class I-based

presentation of exogenous antigen, a poorly described phenomenon called cross-presentation or cross-priming,

has already been described in some cases of tumor cells opsonized by antitumor mAbs [68,69]. Lum and

collaborators have reported an increase in antitumor activity in the endogenous immune cells of patients

treated with ex vivo amplified and activated T-cells retargeted by an anti-CD3 x target. These researchers

hypothesized that secretion of GM-CSF (granulocyte macrophage colony-stimulating factor), RANTES, IFNγ and

IL-2 upon engagement of retargeted T-cells would recruit endogenous T-cells and DCs, leading to vaccination

[11]. Triomabs may have a huge advantage in this respect. These molecules have been demonstrated to

retarget macrophages and DCs toward tumor cells and stimulate these leukocytes via binding to their Fcγ

receptors. In fact, a mouse model study demonstrated that an anti-human EpCAM x murine CD3 triomab could

protect mice against a second tumor challenge that was initiated 144 days after the first challenge. This

protection was proved to be dependent on the presence of the chimeric Fc, as the F(ab')2 fragment of the same

mAb did not lead to long-term protection. Moreover, Linhofer and coworkers observed a strong correlation

between the induction of a humoral immune response with tumor-reactive antibodies and the survival of the

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mice. [10,70]. These results strongly indicate that an induction of long-lasting antitumor immunity was

established in these animals and raises huge expectations for antibody-based cancer therapy.

Conclusion

Through dual binding, the retargeting of effector cells and radiolabeled haptens, a new generation of efficiently

produced bispecific antibodies have the potential to substantially improve antibody-based therapies in the

future, and might even lead to vaccination effects. As several of these molecules are in clinical trials at the

moment and hundreds are in preclinical trials, we should know in the near future whether the beginning of the

end of war against cancer is within reach.

Figure legend

Figure 1. Formats of antibodies.

A conventional antibody (light green for light chains, dark green for heavy chains; blue triangles indicate the

site of glycosylation) and the derived fragments (shaded areas represent the binding sites). Color orange

symbolizes a different specificity. Llama domain antibodies are depicted in mauve or blue.

(Adapted with permission from Chames and Baty © 2009 Chames and Baty)

bsAb bispecific antibody, bsFab bispecific Fab fragment, dAb domain antibody, scFv single-chain Fv fragment

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Figure1

BispecificFab’2(chemicallink)

TandemscFv

scFv

FabFc

SinglechainDiabodyBsFab

bsAb(quadroma)

dAb

Diabody