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A revival of bispecific antibodiesPeter Kufer1,2, Ralf Lutterbuse1 and Patrick A. Baeuerle1

1Micromet AG, Staffelseestr. 2, 81477 Munich, Germany2Institute 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 havebeen 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 Kohler

and Milstein to generate monoclonal antibodies has

nourished the hope for therapeutic breakthroughs in

diseases with high medical needs not served sufficientlyby 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 monoclonalantibodies by chimerization, humanization through

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.Corresponding author: Peter Kufer ([email protected]).

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Although producing small amounts for in vitro studies and

animal models was relatively straightforward, high and

affordable production yields as needed for clinical grade

material required major efforts. Thus, the intense parallel

development of various molecular formats of bispecific

antibodies was largely driven by an attempt to solve

production problems (Figure 1).

The first approach to construct and produce bispecific

antibodies was the quadroma technology [1315]. It is

based on the somatic fusion of two different hybridoma cell

lines expressing murine monoclonal antibodies with the

desired specificities of the bispecific antibody. Because of

the random pairing of two different Ig heavy and light

chains within the resulting hybridhybridoma (or quad-

roma) cell line, up to ten different immunogloblin species

are generated of which only one is the functional bispecific

antibody. The presence of mispaired by-products reduced

production yields significantly and required sophisticatedpurification procedures. A major improvement to the

conventional quadroma approach was the somatic fusion

of a murine and a rat hybridoma cell line expressing

monoclonal antibodies with two IgGsubclasses selected for

their preferential pairing [16]. Bispecific antibodies from

quadroma cell lines still closely resemble conventional

monoclonal antibodies. Besides the dual-specific antigen

binding fragment (Fab) parts, they contain an Fcg part and

can thus be considered trispecific. The additional inter-

action of such trispecific antibodies with Fcg receptors

might have desired as well as undesired biological effects.

To avoid undesired effects, bispecific F(ab0)2 fragments

were prepared by removing the Fcg part through

enzymatic digestion [17]. Other approaches used chemical

conjugation of two different monoclonal antibodies or

smaller antibody fragments [18]. A straightforward

method was the coupling of two parental antibodies with

a hetero-bifunctional crosslinker, but the resulting prep-

arations of bispecific antibodies suffered from a significant

molecular heterogeneity because reaction of the cross-linker with the parental antibodies was not site-directed.

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-

directed manner [19].

Recombinant DNA technology promised to overcome

shortcomings of conventional bispecific antibody pro-

duction. Bispecific antibodies resembling the quadroma-

derived format but consisting of human instead of murine

and/or rat sequences could be designed by the elegant

knobs-into-holes strategy [20]. The mispairing of Ig heavy

chains was reduced in this technology by mutating

selected amino acids forming the interface of the CH3domains in human IgG. At positions within the CH3domain at which the two heavy chains interact directly, an

amino acid with a small side chain (hole) was introduced

into the sequence of one heavy chain and an amino acid

with a large side chain (knob) into that of the other one. As

a result, the more favorable protein interaction between

knobs and holes led to the formation of up to 90% of the

correct bispecific human IgG by transfected mammalian

host cells.

Alternative recombinant strategies focused on smaller

bispecific antibody constructs. A simpler structure and

smaller molecular size should enable bacterial expression

of larger quantities in Escherichia coli. One strategy

made use of the natural ability of certain protein

domains to associate as heterodimers. The leucine

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

heavy chain segments in a Fab-based approach [22].

However, expression in E. coli revealed preferential

formation of stable FosFos and JunJun homodimers.

Therefore, bispecific heterodimers had to be generated

finally in vitro in a second step by mixing the dissociated

halves of homodimers. A similar approach relying on the

heterodimerization of the constant Ig domains Ck

and CH1led to the successful association of two different single-

chain Fv antibody fragments during periplasmicexpression in E. coli [23]. In contrast to the Fos Jun

strategy, no additional in vitro-engineering steps were

required.

Figure 1. Alternative bispecific antibody formats. (a) Quadroma-derived bispecific

antibody, resembling monospecific monoclonal antibodies in structure and size.

(bd) Heterodimeric bispecific antibodies of medium size, comprising constant

immunoglobulin regions and/or heterodimerization domains. (ef) Bispecific anti-

bodies of minimal size, consisting only of variable immunoglobulin regions.Abbreviations: CH13, constant regions of Ig-heavy chain; CL, constant regions of

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-

ors (i.e. gray and purple) represents different binding specificities.

TRENDS in Biotechnology

S-S

VL

VH

CH1

CL

CH2

CH3

CH1Ck

(c)

(a)

(e)

VL

VH

VH

VL VL VH

VL

VH

CH1

CL

VH

VL

Fab

Fc

Quadroma F(ab)2

Heterodimeric scFv Heterodimer ic Fab

Diabody Tandem scFv

VH VL

(b)

(d)

(f)

FosJun

VL

VH

CH1

CL

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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

Vregions. Coexpression of respective VHVL 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 VLVH 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 denaturedprotein fromE. 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 GlySer

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 thathave a key function in disease processes (Figure 2). With

respect to target cells, most approaches selected anti-

bodies for bispecific antibody development recognizing

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 anddendritic 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)2antibodies MDX-210 (Her2/neu CD64), MDX-H210

( humanized MDX-210) and MDX-447 (EGFR CD64)

[40,41], although high doses (20 mg m22) were welltolerated 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 0.11 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

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.

TRENDS in Biotechnology

Triggering receptor

Surfaceantigen

Cytotoxicgranules

Bispecificantibody

Redirectedlysis

Target cell

Cytotoxic effector cell




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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 hybridhybridoma-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 circulatingcytokines, 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 bispecificantibody 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 Hodgkins 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 III 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 3 PR) in a second clinical trial [52].

Therefore, HRS-3/A9 can be regarded as the first clinicalproof 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

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(ab 0)2format as well as to various recombinant diabodies [28,

5358]. 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-cellcytotoxicity (Table 1). So far, indications for clinical efficacy

of CD3-directed bispecific antibodies have come mainly

from locoregional or intraperitoneal treatment of cancer

patients [5962], or from adoptive transfer of Tcells 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 clinicalresponses. 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 TCRCD3 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 clearingpathogens 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

Table 1. Characteristics of bispecific antibodies recruiting cytotoxic effector cells and requirements for redirected target-cell lysis

Triggering molecule CD64 CD89 CD16 CD3

Bispecific antibody format F(ab0)2 F(ab0)2

Quadroma

F(ab0)2Tandem scFv

F(ab0)2Diabody

BiTE

Effector cells Monocytes and neutrophils NK cells T cells

Requirement of pre- or costimulation Yes No Yes Yes NoED50 range [mg ml

21] 0.1 1 0.1 1 0.03 1 0.001 1 10251024

E:T ratio $40:1 100:1 200:1 50:1 $2:1 # 1:10

Refs [44] [46] [49] [28,57] [80]




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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 [32,7678]. This might be explained

by their ability to induce immunological cytolytic synapsesbetween 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 perspectivesTherapeutics based on bispecific antibodies have not yet

yielded the anticipated clinical success. However, this

review shows that further developments are ongoing with

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 tohave 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 discoveredin 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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